A single extra copy of Dscr1 improves survival of mice developing spontaneous lung tumors through suppression of tumor angiogenesis.

The incidence of most solid tumors is remarkably reduced in individuals with Down syndrome. Using mouse models of Down syndrome, we have previously shown that this decrease in tumor incidence is due, in part, to suppression of tumor angiogenesis as a consequence of attenuated calcineurin signaling in endothelial cells. Our prior studies utilized xenografted tumors in a transgenic mouse model with three copies of the Down syndrome critical region-1 (Dscr1) gene, a chromosome 21-encoded endogenous calcineurin inhibitor. These data indicate that upregulated Dscr1 contributes to broad cancer protection by suppressing tumor angiogenesis through inhibiting the calcineurin pathway in the vascular endothelium. However, it still remains to be confirmed whether a single extra copy of Dscr1 is also sufficient to suppress tumor angiogenesis in slow growing spontaneous tumors that more accurately recapitulate molecular features of human malignancies. In this study, utilizing LSL-Kras(G12D) mice, an inducible and autochthonous model of human lung adenocarcinoma, on a Dscr1 transgenic mouse background, we show that a single extra transgenic copy of Dscr1 provides a survival advantage in these mice developing spontaneous lung tumors driven by oncogenic Kras(G12D) without affecting either initiation or progression of spontaneous lung tumors. Furthermore, we show that Dscr1 trisomy significantly reduces microvessel density in lung tumors and thus limits the growth of lung tumors through decreased proliferation and increased apoptosis of lung tumor cells. These data provide evidence that a single extra copy of Dscr1 is sufficient to suppress tumor angiogenesis during spontaneous lung tumorigenesis and further support our hypothesis that suppression of tumor angiogenesis by an additional copy of Dscr1 contributes to the reduced cancer incidence in individuals with Down syndrome and the calcineurin pathway in the tumor vasculature is a potential target for cancer treatment.

Trisomy of the Dscr1 gene suppresses early progression of pancreatic intraepithelial neoplasia driven by oncogenic Kras.

Individuals with Down syndrome exhibit remarkably reduced incidence of most solid tumors including pancreatic cancer. Multiple mechanisms arising from the genetic complexity underlying Down syndrome has been suggested to contribute to such a broad cancer protection. In this study, utilizing a genetically engineered mouse model of pancreatic cancer, we demonstrate that trisomy of the Down syndrome critical region-1 (Dscr1), an endogenous calcineurin inhibitor localized on chromosome 21, suppresses the progression of pancreatic intraepithelial neoplasia-1A (PanIN-1A) to PanIN-1B lesions without affecting the initiation of PanIN lesions mediated by oncogenic Kras(G12D). In addition, we show that Dscr1 trisomy attenuates nuclear localization of nuclear factor of activated T-cells (NFAT) accompanied by upregulation of the p15(Ink4b) tumor suppressor and reduction of cell proliferation in early PanIN lesions. Our data suggest that attenuation of calcineurin-NFAT signaling in neoplastic pancreatic ductal epithelium by a single extra copy of Dscr1 is sufficient to inhibit the progression of early PanIN lesions driven by oncogenic Kras, and thus may be a potential mechanism underlying reduced incidence of pancreatic cancer in Down syndrome individuals.

Depletion of ERK2 but not ERK1 abrogates oncogenic Ras-induced senescence.

In response to oncogenic activation, cells initially undergo proliferation followed by an irreversible growth arrest called oncogene-induced senescence (OIS), an endogenous defense mechanism against tumorigenesis. Oncogenic activation of ERK1/2 is essential for both the initial phase of cellular proliferation as well as subsequent premature senescence, but little is known about the specific contribution of ERK1 versus 2 to OIS. Here we show that depletion of ERK2 but not ERK1 by shRNA knockdown in MEFs leads to continuous proliferation bypassing senescence even in the presence of oncogenic HRAS(V12). Upon depletion of ERK2, induction of both p19(Arf) and p16(Ink4a) was significantly compromised after oncogenic HRAS(V12) expression, attenuating activation of the key tumor suppressors p53 and pRb. Here we demonstrate that ERK2 but not ERK1 indirectly regulates p19(Arf) and p16(Ink4a) both at the transcriptional and translational level. Oncogenic Ras expression after ERK2 knockdown downregulates Fra-1 and c-Jun, components of the activator protein-1 (AP-1) heterodimer essential for transactivation of p19(Arf). Similarly we show a significant decrease in the activation of p38 MAPK and ETS family members which are involved in the induction of p16(Ink4a). The role of ERK2 in translational regulation is observed by the lack of tuberin (TSC2) and p70 ribosomal S6 kinase 1 (p70S6K1) phosphorylation, components of the mTOR pathway, which enhances p19(Arf) mRNA translation during oncogenic Ras-induced senescence. These observations suggest that ERK2 but not ERK1 contributes to upregulation of p19(Arf) and p16(Ink4a) in a transcription- and translation-dependent manner during oncogenic Ras-induced senescence. Taken together, our data indicate that ERK2 is the key ERK isoform mediating the senescence signaling pathway downstream of oncogenic Ras.

Signal transducer and activator of transcription 3 pathway mediates genipin-induced apoptosis in U266 multiple myeloma cells.

It has drawn a lot of attention to target signal transducer and activator of transcription 3 (STAT3) as a potential strategy for cancer therapeutics. Using several myelogenous cell lines, the effect of genipin (an active compound of Gardenia fruit) on the STAT3 pathway and apoptosis was investigated. Genipin suppressed the constitutive STAT3 activation in U266 and U937 cells and stimulated Src homology 2 domain-containing phosphatase 1 (SHP-1), which dephosphorylates and inactivates STAT3. Specifically, genipin blocked STAT3 activation via repressing the activation of c-Src, but not Janus kinase 1 (JAK1). Genipin also downregulated the expression of STAT3 target genes including Bcl-2, Bcl-x(L) , Survivin, Cyclin D1, and VEGF. Conversely, protein tyrosine phosphatase inhibitor pervanadate blocked genipin induced STAT3 inactivation. Using DNA fragmentation or TUNEL assays, we demonstrated the apoptotic effect of genipin on U266, MM.1S, and U937 cells. Furthermore, genipin effectively potentiated the cytotoxic effect of chemotherapeutic agents, such as bortezomib, thalidomide, and paclitaxel in U266 cells. Our data suggest that through regulation of Src and SHP-1, genipin antagonizes STAT3 for the induction of apoptosis in myeloma cells.

Validated quantification for selective cellular uptake of ginsenosides on MCF-7 human breast cancer cells by liquid chromatography-mass spectrometry.

The cellular behavior of ginsenosides on cancer cells has not been measured directly despite their potent anticancer activities and biological actions. A liquid chromatography-mass spectrometry (LC-MS) method was developed to measure the selective cellular uptake of ginsenosides in both cell lysates and culture media. Fifteen ginsenosides were separated within 17 min with good peak shapes using a 2-microm sub-particle size C18 column. Quantification was performed by triple-quadrupole MS with electrospray ionization in negative ion mode. The sample preparation containing the solid-phase extraction was linear (correlation coefficient, r(2) > 0.992) for all analytes, while the limit of quantification ranged from 0.5 to 2.0 ng/mL in both matrices. The assay precision (%CV) and accuracy (%bias) at three different concentrations (5, 20, and 100 ng/mL) were 1.4% to 11.6% and 94.9% to 106.4%, respectively. When this method was used to examine the selective cellular uptake of ginsenosides, the relative non-polar and protopanaxadiol class ginsenosides, such as Rg3, Rk1, Rg5, Rh2, compound-K, and protopanaxadiol (PPD), showed cellular uptake in the MCF-7 cells, but the relative polar and protopanaxatriol class of ginsenosides did not accumulate in the cells. The most non-polar ginsenoside PPD, which is an aglycone of the protopanaxadiol type, resulted in the highest uptake rate. These results show that the different anticancer activities are due to the selective uptake of ginsenosides based on their chemical structures. This LC-MS-based method can be used to estimate the biological activity of ginsenosides on cells from their structural diversity.

Ocimum sanctum induces apoptosis in A549 lung cancer cells and suppresses the in vivo growth of Lewis lung carcinoma cells.

Although Ocimum sanctum has been used extensively for its medicinal values in India and China, its antitumor activity against human nonsmall cell lung carcinoma (NSCLC) A549 cells has not been investigated until now. Therefore, the antitumor mechanism of ethanol extracts of Ocimum sanctum (EEOS) was elucidated in A549 cells in vitro and the Lewis lung carcinoma (LLC) animal model. EEOS exerted cytotoxicity against A549 cells, increased the sub-G1 population and exhibited apoptotic bodies in A549 cells. Furthermore, EEOS cleaved poly(ADP-ribose)polymerase (PARP), released cytochrome C into cytosol and simultaneously activated caspase-9 and -3 proteins. Also, EEOS increased the ratio of proapoptotic protein Bax/antiapoptotic protein Bcl-2 and inhibited the phosphorylation of Akt and extracellular signal regulated kinase (ERK) in A549 cancer cells. In addition, it was found that EEOS can suppress the growth of LLC inoculated onto C57BL/6 mice in a dose-dependent manner. Overall, these results demonstrate that EEOS induces apoptosis in A549 cells via a mitochondria caspase dependent pathway and inhibits the in vivo growth of LLC, suggesting that EEOS can be applied to lung carcinoma as a chemopreventive candidate.