Flavopereirine Inhibits Autophagy via the AKT/p38 MAPK Signaling Pathway in MDA-MB-231 Cells.

Autophagy is a potential target for the treatment of triple negative breast cancer (TNBC). Because of a lack of targeted therapies for TNBC, it is vital to find optimal agents that avoid chemoresistance and metastasis. Flavopereirine has anti-proliferation ability in cancer cells, but whether it regulates autophagy in breast cancer cells remains unclear. A Premo™ Tandem Autophagy Sensor Kit was used to image the stage at which flavopereirine affects autophagy by confocal microscopy. A plasmid that constitutively expresses p-AKT and siRNA targeting p38 mitogen-activated protein kinase (MAPK) was used to confirm the related signaling pathways by Western blot. We found that flavopereirine induced microtubule-associated protein 1 light chain 3 (LC3)-II accumulation in a dose- and time-dependent manner in MDA-MB-231 cells. Confocal florescent images showed that flavopereirine blocked autophagosome fusion with lysosomes. Western blotting showed that flavopereirine directly suppressed p-AKT levels and mammalian target of rapamycin (mTOR) translation. Recovery of AKT phosphorylation decreased the level of p-p38 MAPK and LC3-II, but not mTOR. Moreover, flavopereirine-induced LC3-II accumulation was partially reduced in MDA-MB-231 cells that were transfected with p38 MAPK siRNA. Overall, flavopereirine blocked autophagy via LC3-II accumulation in autophagosomes, which was mediated by the AKT/p38 MAPK signaling pathway.

Arsenic treatment increase Aurora-A overexpression through E2F1 activation in bladder cells.

BACKGROUND: Arsenic is a widely distributed metalloid compound that has biphasic effects on cultured cells. In large doses, arsenic can be toxic enough to trigger cell death. In smaller amounts, non-toxic doses may promote cell proliferation and induces carcinogenesis. Aberration of chromosome is frequently detected in epithelial cells and lymphocytes of individuals from arsenic contaminated areas. Overexpression of Aurora-A, a mitotic kinase, results in chromosomal instability and cell transformation. We have reported that low concentration (≦1 µM) of arsenic treatment increases Aurora-A expression in immortalized bladder urothelial E7 cells. However, how arsenic induces carcinogenesis through Aurora-A activation remaining unclear. METHODS: Bromodeoxyuridine (BrdU) staining, MTT assay, and flow cytometry assay were conducted to determine cell proliferation. Messenger RNA and protein expression levels of Aurora-A were detected by reverse transcriptional-PCR and Western blotting, respectively. Centrosome of cells was observed by immunofluorescent staining. The transcription factor of Aurora-A was investigated by promoter activity, chromosome immunoprecipitation (ChIP), and small interfering RNA (shRNA) assays. Mouse model was utilized to confirm the relationship between arsenic and Aurora-A. RESULTS: We reveal that low dosage of arsenic treatment increased cell proliferation is associated with accumulated cell population at S phase. We also detected increased Aurora-A expression at mRNA and protein levels in immortalized bladder urothelial E7 cells exposed to low doses of arsenic. Arsenic-treated cells displayed increased multiple centrosome which is resulted from overexpressed Aurora-A. Furthermore, the transcription factor, E2F1, is responsible for Aurora-A overexpression after arsenic treatment. We further disclosed that Aurora-A expression and cell proliferation were increased in bladder and uterus tissues of the BALB/c mice after long-term arsenic (1 mg/L) exposure for 2 months. CONCLUSION: We reveal that low dose of arsenic induced cell proliferation is through Aurora-A overexpression, which is transcriptionally regulated by E2F1 both in vitro and in vivo. Our findings disclose a new possibility that arsenic at low concentration activates Aurora-A to induce carcinogenesis.

The correlation between TWIST, E-cadherin, and beta-catenin in human bladder cancer.

PURPOSE: Epithelial-to-mesenchymal transition (EMT)- related factors are known to contribute to the invasion and migration of multiple cancers. However, the expression levels of and the relationship between TWIST, E-cadherin, and beta-catenin in bladder cancer are not yet known. Therefore, this study investigated the relationship between TWIST, E-cadherin, and beta-catenin in tissue specimens and cell lines of bladder cancer. METHODS: Microarrays of bladder cancer tissue and bladder cancer cell lines were used to study the expression levels of TWIST, E-cadherin, and beta-catenin, with disease stage and grade using immunohistochemistry. Moreover, the siRNAs of TWIST, E-cadherin, and beta-catenin were transfected into the bladder cancer cell lines to study any relationship between these factors. RESULTS: The levels of TWIST and beta-catenin were upregulated with increasing grade of malignancy. In contrast, the corresponding results for E-cadherin were just the opposite. Furthermore, inhibition of the expression of TWIST elevated the expression of E-cadherin, but reduced the expression of beta-catenin. However, reduction of beta-catenin by siRNA had no influence on TWIST, but up-regulated the expression of E-cadherin. CONCLUSION: TWIST may act upstream of E-cadherin, which can indirectly regulate the expression levels of beta-catenin. The EMT factors TWIST, E-cadherin, and beta-catenin may be a cluster of biomarkers for the metastatic progression of bladder cancer.

Aurora-A overexpression enhances cell-aggregation of Ha-ras transformants through the MEK/ERK signaling pathway.

BACKGROUND: Overexpression of Aurora-A and mutant Ras (RasV12) together has been detected in human bladder cancer tissue. However, it is not clear whether this phenomenon is a general event or not. Although crosstalk between Aurora-A and Ras signaling pathways has been reported, the role of these two genes acting together in tumorigenesis remains unclear. METHODS: Real-time PCR and sequence analysis were utilized to identify Ha- and Ki-ras mutation (Gly -> Val). Immunohistochemistry staining was used to measure the level of Aurora-A expression in bladder and colon cancer specimens. To reveal the effect of overexpression of the above two genes on cellular responses, mouse NIH3T3 fibroblast derived cell lines over-expressing either RasV12 and wild-type Aurora-A (designated WT) or RasV12 and kinase-inactivated Aurora-A (KD) were established. MTT and focus formation assays were conducted to measure proliferation rate and focus formation capability of the cells. Small interfering RNA, pharmacological inhibitors and dominant negative genes were used to dissect the signaling pathways involved. RESULTS: Overexpression of wild-type Aurora-A and mutation of RasV12 were detected in human bladder and colon cancer tissues. Wild-type Aurora-A induces focus formation and aggregation of the RasV12 transformants. Aurora-A activates Ral A and the phosphorylation of AKT as well as enhances the phosphorylation of MEK, ERK of WT cells. Finally, the Ras/MEK/ERK signaling pathway is responsible for Aurora-A induced aggregation of the RasV12 transformants. CONCLUSION: Wild-type-Aurora-A enhances focus formation and aggregation of the RasV12 transformants and the latter occurs through modulating the Ras/MEK/ERK signaling pathway.

Flavopereirine induces cell cycle arrest and apoptosis via the AKT/p38 MAPK/ERK1/2 signaling pathway in human breast cancer cells.

Breast cancer, which is the most frequently diagnosed cancer, is quite heterogeneous. For breast cancer subtypes lacking targeted therapies, it is vitally essential to find novel agents that prevent chemoresistance and metastatic relapse. Flavopereirine is a ß-carboline alkaloid that has antiplasmodial activity, and its antiproliferative effect in different cancers remains unclear. The effect of flavopereirine on cell cycle arrest and apoptosis signaling in breast cancer cells was analyzed by flow cytometry. An inhibitor and siRNA were used to confirm the related signaling pathways by Western blot analysis. We found that flavopereirine caused G0/G1 phase arrest in MCF-7 cells and S phase arrest in MDA-MB-231 cells. MDA-MB-231 cells were more sensitive to flavopereirine-induced apoptosis. Furthermore, we found that flavopereirine-induced apoptosis was partially reduced in MDA-MB-231 cells treated with an extracellular regulated kinase (ERK) inhibitor and p38 mitogen-activated protein kinase (MAPK) siRNA. Moreover, p38 siRNA treatment simultaneously reduced phosphorylated ERK expression levels. Conversely, the recovered phosphorylation of AKT decreased the levels of p-ERK and p-p38 MAPK. Overall, flavopereirine induces cell cycle arrest and the AKT/p38 MAPK/ERK signaling pathway, which contribute to flavopereirine-induced apoptosis in MDA-MB-231 cells.

Aurora-A overexpression associates with Ha-ras codon-12 mutation and blackfoot disease endemic area in bladder cancer.

Our data revealed that 59.4% of the bladder cancer specimens showed Aurora-A overexpression, of which 31.8% also had Ha-ras codon-12 mutation; 45.5% were from blackfoot-disease endemic areas in which arsenic exposure is a major environment factor associated with various cancer formation. We further demonstrated that arsenic treatment of the immortalized bladder cell line, E7, increased Aurora-A expression. All together, co-existence of Aurora-A overexpression and Ha-ras mutation suggests a possible additively effect on the tumorigenesis of bladder cancer. In addition, Aurora-A overexpression and up-regulated by arsenic exposure opens a new direction for exploring the occurrence of bladder cancer occurrence in Taiwan.

Reversine Induced Multinucleated Cells, Cell Apoptosis and Autophagy in Human Non-Small Cell Lung Cancer Cells.

Reversine, an A3 adenosine receptor antagonist, has been shown to induce differentiated myogenic-lineage committed cells to become multipotent mesenchymal progenitor cells. We and others have reported that reversine has an effect on human tumor suppression. This study revealed anti-tumor effects of reversine on proliferation, apoptosis and autophagy induction in human non-small cell lung cancer cells. Treatment of these cells with reversine suppressed cell growth in a time- and dosage-dependent manner. Moreover, polyploidy occurred after reversine treatment. In addition, caspase-dependent apoptosis and activation of autophagy by reversine in a dosage-dependent manner were also observed. We demonstrated in this study that reversine contributes to growth inhibition, apoptosis and autophagy induction in human lung cancer cells. Therefore, reversine used as a potential therapeutic agent for human lung cancer is worthy of further investigation.

Low concentration of arsenic-induced aberrant mitosis in keratinocytes through E2F1 transcriptionally regulated Aurora-A.

Chronic exposure to low-concentration arsenic promotes cell proliferation and carcinogenesis both in vitro and in vivo. Centrosome amplification, the major cause of chromosome instability, occurs frequently in cancers. Aurora-A is a mitotic kinase and causes centrosome amplification and chromosome instability when overexpressed. Our previous study revealed that low-concentration arsenic induces Aurora-A overexpression in immortalized bladder cells. In this study, we hypothesized that low-concentration arsenic induces aberrant mitosis in keratinocytes due to Aurora-A overexpression. The specimen of Bowen's disease (BD) and squamous cell carcinoma obtained from arseniasis-endemic areas in Taiwan showed Aurora-A overexpression. The mRNA/protein levels and kinase activity of Aurora-A were increased in immortalized keratinocyte HaCaT cells after arsenic treatment at low concentration (< 1µM). Aberrant spindles, multiple centrosomes, and multinucleated cells were detected under fluorescent microscopy in HaCaT cells after arsenic treatment. These findings were associated with increased expression of Aurora-A. We further revealed that Aurora-A was regulated by arsenic-induced transcriptional factor E2F1 as demonstrated by chromosome immunoprecipitation, promoter activity, and small interfering RNA assays. Finally, in arsenic-treated HaCaT cells and in BD, a significant increase of dysfunctional p53 was found, and this event correlated with the increase in expression of Aurora-A. Altogether, our data suggest that low concentration of arsenic induces activation of E2F1-Aurora-A axis and results in aberrant mitosis of keratinocytes. Overexpression of Aurora-A and dysfunctional p53 may act synergistically to trigger skin tumor formation. Our findings suggest that Aurora-A may be a potential target for the prevention and treatment of arsenic-related cancers.

Ha-ras overexpression mediated cell apoptosis in the presence of 5-fluorouracil.

By using a mouse NIH3T3 derivate designed 7-4 harboring the inducible Ha-ras oncogene, we demonstrated the close relationship between Ha-ras expression level and sensitization of 5-flurouracil (5-FU)-treated cells. Further studies revealed that the cells susceptible to 5-FU treatment died of apoptosis, which was demonstrated by caspase-3 activation, loss of mitochondria membrane potential (MMP), and DNA fragmentation. The 7-4 cells coexpressing dominant negative Ras (Ras(Asn17)), dominant negative Raf-1 (Raf-1(CB4)), Bcl-2, or active form of phosphatidylinositol 3-kinase (PI3K) became resistant to 5-FU, and apoptosis was prevented. In contrast, the cells coexpressing dominant negative Rac 1 (Rac1(Asn17)) or dominant negative Rho A (RhoA(Asn19)) showed no change of sensitivity to 5-FU. These results indicate that Ras, Bcl-2, as well as Raf-1 and PI3K pathways play pivotal roles in 5-FU-induced apoptosis under Ha-ras-overexpressed condition. Aberrant levels of cyclin E and p21(Cip/WAF-1) expression as well as Cdc 2 phosphorylation at Tyrosine 15 suggest that perturbation of G1/S and G2/M transitions in cell cycle might be responsible for 5-FU triggered apoptosis. Sensitization of Ha-ras-related cells to 5-FU was also demonstrated in human bladder cancer cells. Through understanding the mechanism of 5-FU induced apoptosis in tumor cells, a new direction toward the treatment of Ha-ras oncogene-related cancers with 5-FU at more optimal dosages is possible and combinational therapy with other drugs that suppress PI3K and Bcl-2 activities can also be considered.