miR‐135b‐ and miR‐146b‐dependent silencing of calcium‐sensing receptor expression in colorectal tumors

Studies have shown that the calcium‐sensing receptor (CaSR) mediates the antitumorigenic effects of calcium against colorectal cancer (CRC). Expression of the CaSR in colorectal tumors is often reduced. We have reported previously that silencing of CaSR in CRC is caused in part by methylation of CaSR promoter 2 and loss of histone acetylation. We investigated the impact of aberrant microRNA expression on loss of CaSR expression. A microarray study in two Caco‐2 subclones (Caco2/AQ and Caco2/15) that have similar genetic background, but different CaSR expression levels (Caco2/AQ expressing more CaSR than Caco2/15), identified 22 differentially expressed microRNAs that potentially target the CaSR. We validated these results by performing gain‐ and loss‐of‐function studies with the top candidates: miR‐9, miR‐27a, miR‐135b, and miR‐146b. Modulation of miR‐135b or miR‐146b expression by mimicking or inhibiting their expression regulated CaSR protein levels in two different colon cancer cell lines: Caco2/AQ (moderate endogenous CaSR expression) and HT29 (low endogenous CaSR levels). Inhibition of miR‐135b and miR‐146b expression led to high CaSR levels and significantly reduced proliferation. In samples of colorectal tumors we observed overexpression of miR‐135b and miR‐146b, and this correlated inversely with CaSR expression (miR‐135b: r = ?0.684, p < 0.001 and miR‐146b: r = ?0.448, p < 0.001), supporting our in vitro findings. We demonstrate that miR‐135b and miR‐146b target the CaSR and reduce its expression in colorectal tumors, reducing the antiproliferative and prodifferentiating actions of calcium. This provides a new approach for finding means to prevent CaSR loss, developing better treatment strategies for CRC.     

Apoptotic signaling in bufalin‐ and cinobufagin‐treated androgen‐dependent and ‐independent human prostate cancer cells

Prostate cancer has its highest incidence in the USA and is becoming a major concern in Asian countries. Bufadienolides are extracts of toxic glands from toads and are used as anticancer agents, mainly on leukemia cells. In the present study, the antiproliferative and apoptotic mechanisms of bufalin and cinobufagin on prostate cancer cells were investigated. Proliferation of LNCaP, DU145, and PC3 cells was measured by 3‐(4,5‐dimethylthiazol‐2‐yle)‐2,5‐diphenyltetrazolium bromide assay and the doubling time (tD) was calculated. Bufalin and cinobufagin caused changes in the tD of three prostate cancer cell lines, which were more significant than that of human mesangial cells. In addition, bufadienolides induced prostate cancer cell apoptosis more significantly than that in breast epithelial cell lines. After treatment, the caspase‐3 activity and protein expression of caspase‐3, ‐8, and ‐9 were elevated. The expression of other apoptotic modulators, including mitochondrial Bax and cytosolic cytochrome c, were also increased. However, expression of p53 was only enhanced in LNCaP cells. Downregulation of p53 by antisense TP53 restored the cell viability suppressed by bufalienolides. Furthermore, the increased expression of Fas was more significant in DU145 and PC3 cells with mutant p53 than in LNCaP cells. Transfection of Fas small interfering RNA restored cell viability in the bufadienolide‐treated cells. These results suggest that bufalin and cinobufagin suppress cell proliferation and cause apoptosis in prostate cancer cells via a sequence of apoptotic modulators, including Bax, cytochrome c, and caspases. The upstream mediators might be p53 and Fas in androgen‐dependent LNCaP cells and Fas in androgen‐independent DU145 and PC3 cells. (Cancer Sci 2008; 99: 2467–2476)     

Non‐invasive estimation of 10B‐4‐borono‐L‐phenylalanine‐derived boron concentration in tumors by PET using 4‐borono‐2‐18F‐fluoro‐phenylalanine

In boron neutron capture therapy (BNCT), ¹⁰B‐4‐borono‐L‐phenylalanine (BPA) is commonly used as a ¹⁰B carrier. PET using 4‐borono‐2‐¹⁸F‐fluoro‐phenylalanine (¹⁸F‐FBPA PET) has been performed to estimate boron concentration and predict the therapeutic effects of BNCT; however, the association between tumor uptake of ¹⁸F‐FBPA and boron concentration in tumors remains unclear. The present study investigated the transport mechanism of ¹⁸F‐FBPA and BPA, and evaluated the utility of ¹⁸F‐FBPA PET in predicting boron concentration in tumors. The transporter assay revealed that 2‐aminobicyclo‐(2.2.1)‐heptane‐2‐carboxylic acid, an inhibitor of the L‐type amino acid transporter, significantly inhibited ¹⁸F‐FBPA and ¹⁴C‐4‐borono‐L‐phenylalanine (¹⁴C‐BPA) uptake in FaDu and LN‐229 human cancer cells. ¹⁸F‐FBPA uptake strongly correlated with ¹⁴C‐BPA uptake in 7 human tumor cell lines (r = .93; P < .01). PET experiments demonstrated that tumor uptake of ¹⁸F‐FBPA was independent of the administration method, and uptake of ¹⁸F‐FBPA by bolus injection correlated well with BPA uptake by continuous intravenous infusion. The results of this study revealed that evaluating tumor uptake of ¹⁸F‐FBPA by PET was useful for estimating ¹⁰B concentration in tumors.     

Interleukin‐10 gene transfer activates interferon‐γ and the interferon‐γ‐inducible genes Gbp‐1/Mag‐1 and Mig‐1 in mammary tumors

Expression of IL‐10 as a transgene inhibits murine mammary tumor growth and metastasis. Using differential display methodology, we sought genes whose expression was modulated by IL‐10. We compared mRNA isolated from parental murine mammary 66.1 tumors, as well as tumors derived from neo^r‐transfected cells and 6 different IL‐10‐expressing cell lines. We identified 2 cDNA products that were up‐regulated in all 6 IL‐10‐expressing tumors in comparison to parental and 66‐neo tumors. One cDNA corresponds to the murine guanylate‐binding protein gene Gbp‐1/Mag‐1. The other cDNA corresponds to the chemokine Mig‐1 (monokine induced by IFN‐γ). Both genes were originally identified in IFN‐γ‐activated macrophages or macrophage cell lines. We now report that cultured mammary epithelial tumor cell lines also express both genes in response to treatment with IFN‐γ and LPS. Furthermore, IFN‐γ mRNA is elevated in IL‐10‐expressing tumors in comparison with parental or neo‐transfected tumors. Thus, high‐level expression of IL‐10 as a transgene results in activation rather than suppression of IFN‐γ as well as 2 IFN‐γ‐inducible genes. Up‐regulation of host IFN‐γ is critical to anti‐tumor activity since IL‐10 no longer inhibits tumor growth in hosts with a deletion in the IFN‐γ gene. Additionally, Gbp‐1/Mag‐1 and Mig‐1 gene induction no longer occur in IFN‐γ mutant mice. Int. J. Cancer 80:624–629, 1999. © 1999 Wiley‐Liss, Inc.     

NY‐ESO‐1 specific antibody and cellular responses in melanoma patients primed with NY‐ESO‐1 protein in ISCOMATRIX and boosted with recombinant NY‐ESO‐1 fowlpox virus

Vaccination strategies based on repeated injections of NY‐ESO‐1 protein formulated in ISCOMATRIX particles (NY‐ESO‐1 ISCOMATRIX) have shown to elicit combined NY‐ESO‐1 specific antibody and T cell responses. However, it remains unclear whether heterologous prime‐boost strategies based on the combination with NY‐ESO‐1 ISCOMATRIX with different NY‐ESO‐1 boosting reagents could be used to increase NY‐ESO‐1 CD8⁺ or CD4⁺ T cell responses. To address this question, we carried out a randomized clinical trial in 39 high‐risk, resected melanoma patients vaccinated with NY‐ESO‐1 ISCOMATRIX, and then boosted with repeated injections of either recombinant fowlpox virus encoding full length NY‐ESO‐1 (rF‐NY‐ESO‐1) (Arm A) or NY‐ESO‐1 ISCOMATRIX alone (Arm B). We have comprehensively analyzed NY‐ESO‐1 specific T cells and B cells response in all patients before and after vaccination for a total of seven time points per patient. NY‐ESO‐1 ISCOMATRIX alone elicited a strong NY‐ESO‐1 specific CD4⁺ T cell and antibody response, which was maintained by both regiments at similar levels. However, CD8⁺ T cell responses were significantly boosted in 3 out of 18 patients in Arm A after the first rF‐NY‐ESO‐1 injection and such responses were maintained until the end of the trial, while no patients in Arm B showed similar CD8⁺ T cell responses. In addition, our results clearly identified immunodominant regions in the NY‐ESO‐1 protein: NY‐ESO‐1_79–102 and NY‐ESO‐1_115–138 for CD4+ T cells and NY‐ESO‐1_85–108 for CD8+ T cells in a large proportion of vaccinated patients. These regions of NY‐ESO‐1 protein should be considered in future clinical trials as immunodominant epitopes.     

μ‐Calpain regulates caspase‐dependent and apoptosis inducing factor‐mediated caspase‐independent apoptotic pathways in cisplatin‐induced apoptosis

Cisplatin, an effective anticancer agent, can induce tumor cell apoptosis via caspase‐dependent and‐independent pathways. However, the precise mechanism that regulates the pathways remains unclear. In this study, we showed that μ‐calpain mediated both caspase‐dependent and‐independent pathways during cisplatin‐induced apoptosis in human lung adenocarcinoma cells. After cisplatin treatment, calpain activation, as measured by a fluorescent substrate, was an early event, taking place well before apoptosis inducing factor (AIF) release and caspase‐9/‐3 activation. Confocal imaging of cells transfected with AIF‐GFP demonstrated that AIF release occurred about 9 hr after cisplatin treatment. The increase of μ‐calpain activity proved to be a crucial event in the apoptotic machinery, as demonstrated by the significant protection of cell death in samples suppressed the endogenous μ‐calpain expression level, as well as cotreated with the calpain inhibitors, calpeptin and PD150606. Inhibition of μ‐calpain not only significantly reduced caspase‐9/‐3 activities but also completely blocked AIF redistribution. Our study also showed that endogenous mitochondrial μ‐calpain could directly induce the truncation and release of AIF, while caspases and cathepsins were not necessary for this process. In conclusion, the study demonstrated that activation of μ‐calpain played an essential role in regulating both caspase‐dependent and AIF‐mediated caspase‐independent apoptotic pathways in cisplatin‐induced apoptosis. © 2009 UICC     

let‐7 and miR‐17‐92: Small‐sized major players in lung cancer development

MicroRNA (miRNA)‐encoding small non‐coding RNA have been recognized as important regulators of a number of biological processes that inhibit the expression of hundreds of genes. Accumulating evidence also indicates the involvement of miRNA alterations in various types of human cancer, including lung cancer, which has long been the leading cause of cancer death in economically well‐developed countries, including Japan. We previously found that downregulation of members of the tumor‐suppressive let‐7 miRNA family and overexpression of the oncogenic miR‐17‐92 miRNA cluster frequently occur in lung cancers, and molecular insight into how these miRNA alterations may contribute to tumor development has been rapidly accumulating. The present review summarizes recent advances in elucidation of the molecular functions of these miRNA in relation to their roles in the pathogenesis of lung cancer. Given the crucial roles of miRNA alterations, additional studies are expected to provide a better understanding of the underlying molecular mechanisms of disease development, as well as a foundation for novel strategies for cancer diagnosis and treatment of this devastating disease. (Cancer Sci 2011; 102: 9–17)     

Attenuation by genistein of sodium‐chloride‐enhanced gastric carcinogenesis induced by N‐methyl‐N′‐nitro‐N‐nitrosoguanidine in Wistar rats

The effects of prolonged administration of genistein, a tyrosine‐kinase inhibitor, on sodium‐chloride‐enhanced induction of gastric carcinogenesis induced by N‐methyl‐N′‐nitro‐N‐nitrosoguanidine, and the labeling and apoptotic indices and vessel counts in the gastric mucosa and gastric cancers, were investigated in Wistar rats. After 25 weeks of the carcinogen treatment, rats were fed chow pellets containing 10% sodium chloride and were given s.c. injections of genistein at dosages of 15 mg/kg or 30 mg/kg body weight every other day. In week 52, the incidence of gastric cancers was significantly greater in rats fed sodium chloride than in untreated control rats. Prolonged administration of genistein at a dosage of 30 mg/kg, but not 15 mg/kg, body weight significantly reduced the incidence of gastric cancers, which was increased by oral treatment with sodium chloride. Genistein at the higher dose significantly decreased the labeling index and vessel counts of the antral mucosa and the gastric cancers (which were increased by treatment with sodium chloride) and significantly increased the apoptotic index of the antral mucosa and the cancers (which was lowered by the treatment with sodium chloride). These findings suggest that genistein attenuates gastric carcinogenesis promoted by sodium chloride, by inducing increased apoptosis and lower cell proliferation and angiogenesis of antral mucosa and gastric cancers. Int. J. Cancer 80:396–399, 1999. © 1999 Wiley‐Liss, Inc.     

Oncogenic microRNA‐27a is a target for anticancer agent methyl 2‐cyano‐3,11‐dioxo‐18β‐olean‐1,12‐dien‐30‐oate in colon cancer cells

Methyl 2‐cyano‐3,11‐dioxo‐18β‐olean‐1,12‐dien‐30‐oate (CDODA‐Me) is a synthetic derivative of glycyrrhetinic acid, a triterpenoid phytochemical found in licorice extracts. CDODA‐Me inhibited growth of RKO and SW480 colon cancer cells and this was accompanied by decreased expression of Sp1, Sp3 and Sp4 protein and mRNA and several Sp‐dependent genes including survivin, vascular endothelial growth factor (VEGF), and VEGF receptor 1 (VEGFR1 or Flt‐1). CDODA‐Me also induced apoptosis, arrested RKO and SW480 cells at G₂/M, and inhibited tumor growth in athymic nude mice bearing RKO cells as xenografts. CDODA‐Me decreased expression of microRNA‐27a (miR‐27a), and this was accompanied by increased expression of 2 miR‐27a‐regulated mRNAs, namely ZBTB10 (an Sp repressor) and Myt‐1 which catalyzes phosphorylation of cdc2 to inhibit progression of cells through G₂/M. Both CDODA‐Me and antisense miR‐27a induced comparable responses in RKO and SW480 cells, suggesting that the potent anticarcinogenic activity of CDODA‐Me is due to repression of oncogenic miR‐27a. © 2009 UICC     

Dual tumor‐suppressors miR‐139‐5p and miR‐139‐3p targeting matrix metalloprotease 11 in bladder cancer

Our recent study of the microRNA (miRNA) expression signature of bladder cancer (BC) by deep‐sequencing revealed that two miRNA, microRNA‐139‐5p/microRNA‐139‐3p were significantly downregulated in BC tissues. The aim of this study was to investigate the functional roles of these miRNA and their modulation of cancer networks in BC cells. Functional assays of BC cells were performed using transfection of mature miRNA or small interfering RNA (siRNA). Genome‐wide gene expression analysis, in silico analysis and dual‐luciferase reporter assays were applied to identify miRNA targets. The associations between the expression of miRNA and its targets and overall survival were estimated by the Kaplan–Meier method. Gain‐of‐function studies showed that miR‐139‐5p and miR‐139‐3p significantly inhibited cell migration and invasion by BC cells. The matrix metalloprotease 11 gene (MMP11) was identified as a direct target of miR‐139‐5p and miR‐139‐3p. Kaplan–Meier survival curves showed that higher expression of MMP11 predicted shorter survival of BC patients (P = 0.029). Downregulated miR‐139‐5p or miR‐139‐3p enhanced BC cell migration and invasion in BC cells. MMP11 was directly regulated by these miRNA and might be a good prognostic marker for survival of BC patients.     

Benefits of catch‐up in vaccination against human papillomavirus in medium‐ and low‐income countries

Human papillomavirus (HPV) vaccination of a birth cohort of girls in the 9–13 age range is recommended as a priority, but decreases in HPV vaccine cost may make catch‐up of a few additional cohorts more attractive not only in high‐income countries. We assessed the reduction in HPV16 and 18 infections that could be achieved in a medium‐ (Poland) and a low‐income (Guinea) country by adding one‐time catch‐up of 12‐ to 19‐year‐old girls to the vaccination of 11‐year‐old girls. According to our ad hoc adapted dynamic model of HPV infection transmission, the addition of catch‐up was estimated to bring forward the 50% reduction of HPV16/18 prevalence due to vaccination in women ≤35 by as much as 5 years. Catch‐up of 12‐ to 15‐year olds reduced the cumulative probability of HPV16/18 infections by age 35 in the relevant cohorts by about 30% in both countries. Catch‐up of 16‐ to 19‐year‐old girls added little. Regardless of the chosen catch‐up strategy, 16 to 20% of HPV16/18 prevention from vaccination was attributable to herd immunity. Assuming a sufficiently low vaccine cost, the addition of a catch‐up round is, therefore, worth considering in medium/low‐income countries to extend vaccine benefits to less young adolescent girls whose future access to cervical screening is uncertain.