Mohammed Mohammed Ibrahim

Genistein decreases the breast cancer stem-like cell population through Hedgehog pathway

IntroductionThe existence of breast cancer stem-like cells (BCSCs) has profound implications for cancer prevention. Genistein, a predominant isoflavone found in soy products, has multiple robust anti-tumor effects in various cancers, especially in the breast and prostate cancer. In this study, we aimed to evaluate genistein inhibition of BCSCs and its potential mechanism by culturing MCF-7 breast cancer cells and implanting these cells into nude mice.MethodsCell counting, colony formation and cell apoptosis analysis were used to evaluate the effect of genistein on breast cancer cells’ growth, proliferation and apoptosis. We then used mammosphere formation assay and CD44CD24 staining to evaluate the effect of genistein on BCSCs in vitro. A nude mice xenograft model was employed to determine whether genistein could target BCSCs in vivo, as assessed by real-time polymerase chain reaction (PCR) and immunohistochemical staining. The potential mechanism was investigated utilizing real-time PCR, western blotting analysis and immunohistochemical staining.ResultsGenistein inhibited the MCF-7 breast cancer cells’ growth and proliferation and promoted apoptosis. Both in vitro and in vivo genistein decreased breast cancer stem cells, and inhibited breast cancer stem-like cells through down-regulation of the Hedgehog-Gli1 Signaling Pathway.ConclusionsWe demonstrated for the first time that genistein inhibits BCSCs by down-regulating Hedgehog-Gli1 signaling pathway. These findings provide support and rationale for investigating the clinical application of genistein in treating breast cancer, and specifically by targeting breast cancer stem cells.

Annexin A7 gene is an important factor in the lymphatic metastasis of tumors

In the tumor malignancy progression, lymph node metastasis (LNM) is recognized as an important factor. In this study, RNA interference (RNAi) was employed to down-regulate ANXA7 gene in Hca-F cells, a hepatocarcinoma cell line with high LNM rate. There was no significant effect on cell proliferation ability, but cell division, motility, and invasion abilities were markedly inhibited. By contrast, up-regulating the expression of ANXA7 gene in Hca-P cells with lower LNM rate, cell migration ability was improved and the percentage of cells in S phase was significantly decreased in vitro. Here, we reported that the expression of Ech1, GSN and JNK1 genes, which were relevant to tumor lymphatic metastasis, had been inhibited due to down-regulation ANXA7 gene and promoted due to up-regulation ANXA7 gene by western blot analysis. These results indicated that ANXA7 is a critical factor in the development of lymphatic metastasis in hepatocarcinoma progression.

Annexin A7 and its binding protein galectin-3 influence mouse hepatocellular carcinoma cell line in vitro

Lymph node metastasis is recognized as an important mode of liver cancer metastasis. Our previous study has built two hepatocarcinoma cell lines, Hca-F with high (75%) and Hca-P with low (25%) incidences of lymph node metastasis, and has indicated that annexin A7 is an important factor in the lymphatic metastasis of tumors. There is evidence that galectin-3 is the binding protein of annexin A7 and works in protein complexes. Our current study shows that both annexin A7 and galectin-3 express higher in Hca-F than Hca-P. Annexin A7 was successfully down-regulated in Hca-P by RNA interference, and this resulted in concomitant reduction of galactin 3 expression in annexin A7 down regulated compared to the control and N-control cells. Using CCK-8 assay, the expression level of annexin A7 and galectin-3 were found to have correlation with the proliferation ability; Transwell assay showed annexin A7 and galectin-3 are involved in cell migration and invasion regulation in mouse hepatocellular carcinoma cell lines, immunofluorescence assay indicate annexin A7 and galectin-3 were co-located annexin A7 and galectin-3 played roles in DNA damage and cell proliferation cycle checkpoint arrest pathway. Those phenomena indicated that annexin A7 influences lymphatic metastasis of tumors by interacting with galectin-3 through the regulation of tumor cell proliferation, attachment, migration and invasion.

Down-regulation of ANXA7 decreases metastatic potential of human hepatocellular carcinoma cells in vitro

We report for the first time the influence of ANXA7 gene on human hepatocellular carcinoma cells (HCC). We down-regulated ANXA7 in human HCC cell line (HepG2) using siRNA method. By Western Blot analysis, we confirmed about 70% down-regulation of the gene in the shRNA-ANXA7 transfected cells (shRNA-ANXA7-HepG2) compared to the non-specific sequence shRNA transfected cells (control-shRNA-HepG2) and the un-manipulated-HepG2 cells. We used CCK-8 cell proliferation kit and observed about 65% reduction in the shRNA-ANXA7-HepG2 cells where the two controls exhibited comparable cell proliferation rates. Also, by using PI staining followed by flow cytometry, we noticed a cell cycle arrest at G0/G1 with more than one fold reduction of shRNA-ANXA7-HepG2 cell population in the S-phase of the cell cycle. Also of particular note was a significant aneuploidy in the controls compared to zero aneuploidy in the ANXA7 down-regulated cells. Migration of the cells was detected using Boydens transwell chamber and scratch wound healing assay which showed 50% and 30% respective reductions in shRNA-ANXA7-HepG2 cells migration. Furthermore, the control-shRNA-HepG2 cells and the un-manipulated-HepG2 cells invaded through the ECM-coated transwell plates two times more than the shRNA-ANXA7-HepG2 cells. We have found ANXA7 to be functioning like a tumour promoter in HepG2 human hepatocellular carcinoma cells and could have a potential as a therapeutic window into the management of liver cancer.

Ech1 is a potent suppressor of lymphatic metastasis in hepatocarcinoma.

We have previously demonstrated that Ech1 is involved in the lymphatic metastasis of tumors in vitro. Here, we gain an insight into the role that Ech1 is playing in Hca-F cell. The expression of Annexin A7, Gelsolin and Clic1 genes, which were also relevant to tumor lymphatic metastasis, had been inhibited due to downregulation Ech1 gene by Western blot analysis. And downregulated of Ech1 inhibits the metastasic capability of Hca-F cells to peripheral lymph nodes in vivo. Our work indicates although the involvement of Ech1 in tumor metastasis development and progression, but the subcellular location of Ech1 has not much contribution to that.

Enoyl-coenzyme A hydratase in cancer.

Enoyl-CoA hydratase (Ech) catalyzes the second step in the physiologically important beta-oxidation pathway of fatty acid metabolism. The enzyme was reported to be associated with the progression, metastasis and drug resistance of cancers. It might function as a tumor promoter or a tumor suppressor for certain cancers depending on the particular type or stage of tumor cells/tissues. In this review, Echs association with malignant tumors as well as its potential mechanisms is discussed and summarized. The enzyme might be useful in the diagnosis, treatment and prognosis determination of certain tumors.

Evaluation of Annexin A7, Galectin-3 and Gelsolin as possible biomarkers of hepatocarcinoma lymphatic metastasis.

We have previously demonstrated that Annexin A7 is involved in the lymphatic metastasis of hepatocarcinoma in vitro. The expression of Galectin-3 and Gelsolin, which were also relevant to tumor lymphatic metastasis, had shown the same tendency concordantly with the expression of Annexin A7 alteration by qRT-PCR and Western blot analysis. Here, we gain an insight into the role that Annexin A7 is playing in Hca-P, PAnxa7-upregulated and PAnxa7-downregulated cells in vivo. Then, Hca-P, PAnxa7-upregulated and PAnxa7-downregulated cells were injected into a mouse footpad to establish primary tumors in mice. On the fourth week after HCC cells inoculation, the mice were sacrificed for inspection the expression of Annexin A7, Galectin-3 and Gelsolin in primary tumors and in serum. Our work indicates that Annexin A7 and Gelsolin are both valuable in tumors and in serum evaluating lymph node metastasis in mice with hepatocarcinoma; Galectin-3 in tumors is significant but no much contribution in serum.

Guanine nucleotide-binding protein subunit beta-2-like 1, a new Annexin A7 interacting protein.

We report for the first time that Guanine nucleotide-binding protein subunit beta-2-like 1 (RACK1) formed a complex with Annexin A7. Hca-F and Hca-P are a pair of syngeneic mouse hepatocarcinoma cell lines established and maintained in our laboratory. Our previous study showed that both Annexin A7 and RACK1 were expressed higher in Hca-F (lymph node metastasis >70%) than Hca-P (lymph node metastasis <30%). Suppression of Annexin A7 expression in Hca-F cells induced decreased migration and invasion ability. In this study, knockdown of RACK1 by RNA interference (RNAi) had the same impact on metastasis potential of Hca-F cells as Annexin A7 down-regulation. Furthermore, by co-immunoprecipitation and double immunofluorescence confocal imaging, we found that RACK1 was in complex with Annexin A7 in control cells, but not in the RACK1-down-regulated cells, indicating the abolishment of RACK1-Annexin A7 interaction in Hca-F cells by RACK1 RNAi. Taken together, these results suggest that RACK1-Annexin A7 interaction may be one of the means by which RACK1 and Annexin A7 influence the metastasis potential of mouse hepatocarcinoma cells in vitro.

UCP2 protects against amyloid beta toxicity and oxidative stress in primary neuronal culture

AD is a common neurodegenerative disease characterized by aggregated amyloid-beta (Aβ) peptide, and oxidative stress, while uncoupling protein 2 (UCP2) is a member of the anion carrier family, predicted the existence of a protein-regulated proton leak with the main purpose of controlling mitochondrial oxidative stress, reduce the generation of superoxide anion. we use the primary hippocampal neurons and add the different doses of Aβ1-40, then observe the change of UCP2 at different concentrations of Aβ, activity of LDH and the content of NO. Our results provide novel insight that UCP2 may protect hippocampal neurons exposed to amyloid β protein through decreasing ROS production. 20μmol/L Aβ1-40 significantly increased the activity of LDH and the content of NO. According to the correlation analysis, NO was significantly correlation with LDH, and UCP2 was significantly correlation with NO. These results suggest the potential of UCP2 as a therapeutic candidate for treating neurodegenerative diseases such as AD.