Silencing of miR-21 sensitizes CML CD34+ stem/progenitor cells to imatinib-induced apoptosis by blocking PI3K/AKT pathway.

BCR-ABL tyrosine kinase inhibitor imatinib fails to eradicate leukemia stem cells (LSCs), the underlying mechanisms maintaining CML LSCs remain poorly understood. Here, we showed that transient inhibition of miR-21 by antagomiR-21 markedly increased imatinib-induced apoptosis in CML, but not normal CD34+ stem/progenitor cells. Furthermore, PI3K inhibitors also significantly sensitized CML CD34+ cells to imatinib-induced apoptosis. MiR-21 or PI3K inhibitor in combination with imatinib treatment significantly decreased AKT phosphorylation and c-Myc expression than either agent did alone, but did not affect Bim and Bcl-6 expresssion. These findings indicate that miR-21 is required for maintaining the imatinib-resistant phenotype of CML CD34+ cells through PI3K/AKT signaling pathway, thus providing the basis for a promising therapeutic approach to eliminate CML LSCs.

Targeting miR-21 sensitizes Ph+ ALL Sup-b15 cells to imatinib-induced apoptosis through upregulation of PTEN.

Philadelphia chromosome positive (Ph+) acute lymphoblastic leukemia (ALL) cells are insensitive to BCR-ABL tyrosine kinase inhibitor imatinib, the underlying mechanisms remain largely unknown. Here, we showed that imatinib treatment induced significant upregulation of miR-21 and downregulation of PTEN in Ph+ ALL cell line Sup-b15. Transient inhibition of miR-21 resulted in increased apoptosis, PTEN upregulation and AKT dephosphorylation, whereas ectopic overexpression of miR-21 further conferred imatinib resistance. Furthermore, knockdown of PTEN protected the cells from imatinib-induced apoptosis achieved by inhibition of miR-21. Additionally, PI3K inhibitors also notably enhanced the effects of imatinib on Sup-b15 cells and primary Ph+ ALL cells similar to miR-21 inhibitor. Therefore, miR-21 contributes to imatinib resistance in Ph+ ALL cells and antagonizing miR-21 demonstrates therapeutic potential by sensitizing the malignancy to imatinib therapy.

[Gleevec induces apoptosis in K562 cells through activating caspase-3].

The present study is to elucidate the mechanisms underlying Gleevec-induced apoptosis of chronic myeloid leukemia (CML) K562 cells in vitro. The apoptotic cell death and cell cycle distribution after Gleevec treatment and the effect of PDCD4 siRNA on Gleevec-induced apoptosis of K562 cells were analyzed by flow cytometry. The effect of Gleevec on p-Crkl, caspase-3, PARP and PDCD4 protein levels, and the knockdown efficacy of PDCD4 siRNA were detected by Western blotting. The results showed that Gleevec dramatically suppressed the phosphorylation level of Crkl in a dose-dependent manner and induced significant apoptosis and G0/G1 cell cycle arrest of K562 cells in time- and dose-dependent manners. In addition, Gleevec activated caspase-3 and its downstream substrates PARP, and the caspase pan inhibitor Z-VAD-FMK (50 micromol x L(-1)) markedly reduced Gleevec-induced apoptosis from 47.97% +/- 10.56% to 31.05% +/- 9.206% (P < 0.05). Moreover, Gleevec significantly increased the protein expression of programmed cell death 4 (PDCD4). PDCD4 knockdown by siRNA reduced Gleevec-induced apoptosis from 46.97% +/- 14.32% to 42.8% +/- 11.43%. In summary, Gleevec induced apoptosis in K562 cells via caspase-3 activation.

Cecropin suppresses human hepatocellular carcinoma BEL- 7402 cell growth and survival in vivo without side-toxicity.

Conventional chemotherapy against hepatocellular carcinoma typically causes various side effects. Our previous study showed that cecropin of Musca domestica can induce apoptosis in human hepatocellular carcinoma BEL-7402 cells in vitro. However, whether cecropin inhibits BEL-7402 cell in vivo and the question of possible side effects remained undentified. The present study confirmed tumor-inhibitory effects of cecropin in vivo, and furthermore strongly suggested that cecropin cytotoxicity in BEL-7402 cells in vivo may be mainly derived from its pro-apoptotic action. Specifically, we found that cecropin exerted no obvious side effects in tumor-bearing mice as it had no significant hematoxicity as well as visceral toxicity. Therefore, cecropin may be a potential candidate for further investigation as an antitumor agent against hepatocellular carcinoma.

Effects of Musca domestica cecropin on the adhesion and migration of human hepatocellular carcinoma BEL-7402 cells.

This study was designed to explore the effects of Musca domestica antimicrobial peptides cecropin on the adhesion and migration of human hepatocellular carcinoma BEL-7402 cells. The adhesive and migratory capacities were determined by adhesion assay and transwell assay, respectively. The changes in microvilli of tumor cells were determined by scanning electron microscopy (SEM). Western blotting and quantitative polymerase chain reaction (qPCR) were carried out to determine the expression levels of proteins related to adhesion and migration, such as matrix metalloproteinase-2 (MMP2), tissue inhibitors of metalloproteinase-2 (TIMP2), and epithelial cadherin (E-cadherin). We found that Musca domestica cecropin inhibited the adhesion and migration of BEL-7402 cells, which also displayed curling microvilli, increased ball structures on cell surface, gradually broken connections between tumor cells, and even disappeared microvilli on some cells. The expression of MMP2 was significantly reduced after cecropin treatment, while the levels of TIMP2 and E-cadherin were significantly increased. These results suggest that Musca domestica cecropin inhibits the adhesion and migration of human hepatocellular carcinoma BEL-7402 cells by destroying the microvilli of tumor cells and changing the expression of MMP2, TIMP2 and E-cadherin.

Curcumin induces FasL-related apoptosis through p38 activation in human hepatocellular carcinoma Huh7 cells.

AIM: The aim of this study is to explore the underlying molecular mechanism of curcumin-induced apoptosis in human hepatocellular carcinoma (HCC) Huh7 cells. MAIN METHODS: Fas and FasL mRNA expression was analyzed by reverse transcription PCR. Western blot was applied to detect the protein expression of Bcl-2 family members, MAPK family members, c-Jun, c-Fos, ATF-2, caspase-3, PARP, TNF receptor family members and the respective ligands. Apoptotic cells were assayed with annexin V/PI double staining and flow cytometry. KEY FINDINGS: Curcumin treatment resulted in a fast and significant increase of Fas and Fas ligand (FasL) along with activation of caspase-3 and cleavage of PARP in Huh7 cells. Inhibition of caspase-3 activity by the specific inhibitor Z-DEVD-FMK rescued Huh7 cells from curcumin-induced apoptosis. Neutralization of FasL significantly protected the cells from curcumin-induced caspase-3 activation and apoptosis in a dose-dependent manner. Moreover, p38 was rapidly activated in response to curcumin, and inactivation of p38 by pharmacologic inhibitor SB203580 dramatically suppressed curcumin-induced FasL expression and apoptosis. SIGNIFICANCE: Our results demonstrated that curcumin induces apoptosis through p38-denpendent up-regulation of FasL in Huh7 cells.