Malignant phyllodes tumors display mesenchymal stem cell features and aldehyde dehydrogenase/disialoganglioside identify their tumor stem cells.

IntroductionAlthough breast phyllodes tumors are rare, there is no effective therapy other than surgery. Little is known about their tumor biology. A malignant phyllodes tumor contains heterologous stromal elements, and can transform into rhabdomyosarcoma, liposarcoma and osteosarcoma. These versatile properties prompted us to explore their possible relationship to mesenchymal stem cells (MSCs) and to search for the presence of cancer stem cells (CSCs) in phyllodes tumors.MethodsParaffin sections of malignant phyllodes tumors were examined for various markers by immunohistochemical staining. Xenografts of human primary phyllodes tumors were established by injecting freshly isolated tumor cells into the mammary fat pad of non-obese diabetic-severe combined immunodeficient (NOD-SCID) mice. To search for CSCs, xenografted tumor cells were sorted into various subpopulations by flow cytometry and examined for their in vitro mammosphere forming capacity, in vivo tumorigenicity in NOD-SCID mice and their ability to undergo differentiation.ResultsImmunohistochemical analysis revealed the expression of the following 10 markers: CD44, CD29, CD106, CD166, CD105, CD90, disialoganglioside (GD2), CD117, Aldehyde dehydrogenase 1 (ALDH), and Oct-4, and 7 clinically relevant markers (CD10, CD34, p53, p63, Ki-67, Bcl-2, vimentin, and Globo H) in all 51 malignant phyllodes tumors examined, albeit to different extents. Four xenografts were successfully established from human primary phyllodes tumors. In vitro, ALDH+ cells sorted from xenografts displayed approximately 10-fold greater mammosphere-forming capacity than ALDH- cells. GD2+ cells showed a 3.9-fold greater capacity than GD2- cells. ALDH+/GD2+cells displayed 12.8-fold greater mammosphere forming ability than ALDH-/GD2- cells. In vivo, the tumor-initiating frequency of ALDH+/GD2+ cells were up to 33-fold higher than that of ALDH+ cells, with as few as 50 ALDH+/GD2+ cells being sufficient for engraftment. Moreover, we provided the first evidence for the induction of ALDH+/GD2+ cells to differentiate into neural cells of various lineages, along with the observation of neural differentiation in clinical specimens and xenografts of malignant phyllodes tumors. ALDH+ or ALDH+/GD2+ cells could also be induced to differentiate into adipocytes, osteocytes or chondrocytes.ConclusionsOur findings revealed that malignant phyllodes tumors possessed many characteristics of MSC, and their CSCs were enriched in ALDH+ and ALDH+/GD2+ subpopulations

Malignant phyllodes tumors display mesenchymal stem cell features and aldehyde dehydrogenase/disialoganglioside identify their tumor stem cells

INTRODUCTION: Although breast phyllodes tumors are rare, there is no effective therapy other than surgery. Little is known about their tumor biology. A malignant phyllodes tumor contains heterologous stromal elements, and can transform into rhabdomyosarcoma, liposarcoma and osteosarcoma. These versatile properties prompted us to explore their possible relationship to mesenchymal stem cells (MSCs) and to search for the presence of cancer stem cells (CSCs) in phyllodes tumors. METHODS: Paraffin sections of malignant phyllodes tumors were examined for various markers by immunohistochemical staining. Xenografts of human primary phyllodes tumors were established by injecting freshly isolated tumor cells into the mammary fat pad of non-obese diabetic-severe combined immunodeficient (NOD-SCID) mice. To search for CSCs, xenografted tumor cells were sorted into various subpopulations by flow cytometry and examined for their in vitro mammosphere forming capacity, in vivo tumorigenicity in NOD-SCID mice and their ability to undergo differentiation. RESULTS: Immunohistochemical analysis revealed the expression of the following 10 markers: CD44, CD29, CD106, CD166, CD105, CD90, disialoganglioside (GD2), CD117, Aldehyde dehydrogenase 1 (ALDH), and Oct-4, and 7 clinically relevant markers (CD10, CD34, p53, p63, Ki-67, Bcl-2, vimentin, and Globo H) in all 51 malignant phyllodes tumors examined, albeit to different extents. Four xenografts were successfully established from human primary phyllodes tumors. In vitro, ALDH(+) cells sorted from xenografts displayed approximately 10-fold greater mammosphere-forming capacity than ALDH(-) cells. GD2(+) cells showed a 3.9-fold greater capacity than GD2(-) cells. ALDH(+)/GD2(+)cells displayed 12.8-fold greater mammosphere forming ability than ALDH(-)/GD2(-) cells. In vivo, the tumor-initiating frequency of ALDH(+)/GD2(+) cells were up to 33-fold higher than that of ALDH(+) cells, with as few as 50 ALDH(+)/GD2(+) cells being sufficient for engraftment. Moreover, we provided the first evidence for the induction of ALDH(+)/GD2(+) cells to differentiate into neural cells of various lineages, along with the observation of neural differentiation in clinical specimens and xenografts of malignant phyllodes tumors. ALDH(+) or ALDH(+)/GD2(+) cells could also be induced to differentiate into adipocytes, osteocytes or chondrocytes. CONCLUSIONS: Our findings revealed that malignant phyllodes tumors possessed many characteristics of MSC, and their CSCs were enriched in ALDH(+) and ALDH(+)/GD2(+) subpopulations

Berberine enhances inhibition of glioma tumor cell migration and invasiveness mediated by arsenic trioxide

<p>Abstract</p> <p>Background</p> <p>Arsenic trioxide (As₂O₃) exhibits promising anticarcinogenic activity in acute promyelocytic leukemic patients and induces apoptosis in various tumor cells <it>in vitro</it>. Here, we investigated the effect of the natural alkaloid berberine on As₂O₃-mediated inhibition of cancer cell migration using rat and human glioma cell lines.</p> <p>Methods</p> <p>The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was used to determine the viability of rat C6 and human U-87 glioma cells after treatment with As₂O₃or berberine, and after co-treatment with As₂O₃and berberine. The wound scratch and Boyden chamber assays were applied to determine the effect of As₂O₃and berberine on the migration capacity and invasiveness of glioma cancer cells. Zymography and Western blot analyses provided information on the effect of As₂O₃and berberine on the intracellular translocation and activation of protein kinase C (PKC), and some PKC-related downstream factors. Most assays were performed three times, independently, and data were analyzed using ANOVA.</p> <p>Results</p> <p>The cell viability studies demonstrated that berberine enhances As₂O₃-mediated inhibition of glioma cell growth after 24 h incubation. Untreated control cells formed a confluent layer, the formation of which was inhibited upon incubation with 5 μM As₂O₃. The latter effect was even more pronounced in the presence of 10 μM berberine. The As₂O₃-mediated reduction in motility and invasion of glioma cells was enhanced upon co-treatment with berberine. Furthermore, it has been reported that PKC isoforms influence the morphology of the actin cytoskeleton, as well as the activation of metalloproteases MT1-MMP and MMP-2, reported to be involved in cancer cell migration. Treatment of glioma cells with As₂O₃and berberine significantly decreased the activation of PKC α and ε and led to actin cytoskeleton rearrangements. The levels of two downstream transcription factors, myc and jun, and MT1-MMP and MMP-2 were also significantly reduced.</p> <p>Conclusion</p> <p>Upon co-treatment of glioma cells with As₂O₃and berberine, cancer cell metastasis can be significantly inhibited, most likely by blocking the PKC-mediated signaling pathway involved in cancer cell migration. This study is potentially interesting for the development of novel chemotherapeutic approaches in the treatment of malignant gliomas and cancer development in general.</p

Hibiscus sabdariffa Leaf Extract Inhibits Human Prostate Cancer Cell Invasion via Down-Regulation of Akt/NF-kB/MMP-9 Pathway

Hibiscus sabdariffa leaf has been previously shown to possess hypoglycemic, hypolipidemic, and antioxidant effects, and induce tumor cell apoptosis. However, the molecular mechanisms involved in the anticancer activity of H. sabdariffa leaf extract (HLE) are poorly understood. The object of the study was to examine the anti-invasive potential of HLE. First, HLE was demonstrated to be rich in polyphenols. The results of wound-healing assay and in vitro transwell assay revealed that HLE dose-dependently inhibited the migration and invasion of human prostate cancer LNCaP (lymph node carcinoma of the prostate) cells under non-cytotoxic concentrations. Our results further showed that HLE exerted an inhibitory effect on the activity and expressions of matrix metalloproteinase-9 (MMP-9). The HLE-inhibited MMP-9 expression appeared to be a consequence of nuclear factor-kappaB (NF-κB) inactivation because its DNA-binding activity was suppressed by HLE. Molecular data showed all these influences of HLE might be mediated via inhibition of protein kinase B (PKB, also known as Akt)/NF-kB/MMP-9 cascade pathway, as demonstrated by the transfection of Akt1 overexpression vector. Finally, the inhibitory effect of HLE was proven by its inhibition on the growth of LNCaP cells and the expressions of metastasis-related molecular proteins in vivo. These findings suggested that the inhibition of MMP-9 expression by HLE may act through the suppression of the Akt/NF-kB signaling pathway, which in turn led to the reduced invasiveness of the cancer cells

C6 glioma cells were incubated for 12 and 24 h with 10 μM berberine (Ber) or 5 μM AsO(As), and with 10 μM berberine and 5 μM AsO(A Ber) (A, B)

Cytosolic (B) and membrane fractions (A) were evaluated for the presence of PKCα and PKCε by Western blotting. Control cells were not treated in the presence of 10% Fetal Calf Serum (FCS) (N10). Cells that were not exposed to agents were used as negative control in the presence of 1% FCS (N1). The PKC levels in the respective fractions were quantified and normalized taking the β-actin value as a loading control as presented in the graph. Each value is the relative ratio of PKCs membrane to cytosol fraction (presumably, the ratio of untreated control is 1). In addition, the nuclear protein fractions were evaluated for the presence of myc and c-jun, two transcription factors that act downstream of PKC, by Western blotting (C). The quantitative data were presented as three repeats from one independent experiment and indications are in panel A. The intensity of the protein bands was quantified and the level relative to the N10 control band was presented (fold).<p><b>Copyright information:</b></p><p>Taken from "Berberine enhances inhibition of glioma tumor cell migration and invasiveness mediated by arsenic trioxide"</p><p>http://www.biomedcentral.com/1471-2407/8/58</p><p>BMC Cancer 2008;8():58-58.</p><p>Published online 25 Feb 2008</p><p>PMCID:PMC2275285.</p><p></p

C6 glioma cells were incubated for 24 h with 10 μM berberine (Ber) or 5 μM AsO(As), and with 10 μM berberine and 5 μM AsO(A Ber)

Total cell lysates were prepared and subjected to Western blot analysis. Protein levels of phosphorylated ERK1/ERK2 and non-phosphorylated ERK1/ERK2, and ODC, MT1-MMP and MMP2 were detected using the respective monoclonal antibodies (A). The condition serum free media were collected for gelatin zymography analysis from C6 glioma and U-87 cells. Determined activities of these proteins were subsequently quantified by densitometric analysis with the value of controls set at 100% as shown in the graph. The quantitative data were presented as the mean of three repeats from one independent experiment. Other data in this figure is presented as mean ± SD of three independent experiments. * and ** indicate means that are significantly different when compared to the control group of C6 with < 0.05 and < 0.01, respectively. and indicate means that are significantly different when compared to the control group of U-87 with < 0.05 and < 0.01, respectively.<p><b>Copyright information:</b></p><p>Taken from "Berberine enhances inhibition of glioma tumor cell migration and invasiveness mediated by arsenic trioxide"</p><p>http://www.biomedcentral.com/1471-2407/8/58</p><p>BMC Cancer 2008;8():58-58.</p><p>Published online 25 Feb 2008</p><p>PMCID:PMC2275285.</p><p></p

C6 glioma cells were incubated with 5 μM AsOor 10 μM berberine alone and with 5 μM AsOand 10 μM berberine for 4, 12, and 24 h and the migration was visualized as described in Methods (A)

The percentage of surface area filled by the C6 cells was subsequently quantified by densitometric analyses relative to that of the control which was set at 100% as shown in the graph (B). Data are presented as means ± SD based on three independent experiments. * and ** indicate means that are significantly different when compared to the control group of 12 h incubation with < 0.05 and < 0.01, respectively. and indicate means that are significantly different when compared to the control group of 24 h incubation with < 0.05 and < 0.01, respectively.<p><b>Copyright information:</b></p><p>Taken from "Berberine enhances inhibition of glioma tumor cell migration and invasiveness mediated by arsenic trioxide"</p><p>http://www.biomedcentral.com/1471-2407/8/58</p><p>BMC Cancer 2008;8():58-58.</p><p>Published online 25 Feb 2008</p><p>PMCID:PMC2275285.</p><p></p

C6 glioma cells were incubated with 5 μM AsOor 10 μM berberine and co-treated with 5 μM and 10 μM berberine for 24 h

Actin rearrangements were visualized by immunolocalization using anti-F-actin antibodies as described in Methods. Co-treatment with berberine and AsOresulted in actin () impolarization at the edges of the cell. Actin ruffing at the edges of the elongated cells was also observed and indicative of increased migration.<p><b>Copyright information:</b></p><p>Taken from "Berberine enhances inhibition of glioma tumor cell migration and invasiveness mediated by arsenic trioxide"</p><p>http://www.biomedcentral.com/1471-2407/8/58</p><p>BMC Cancer 2008;8():58-58.</p><p>Published online 25 Feb 2008</p><p>PMCID:PMC2275285.</p><p></p