Deding Tao

Fibroblast-Derived Exosomes Contribute to Chemoresistance through Priming Cancer Stem Cells in Colorectal Cancer.

Chemotherapy resistance observed in patients with colorectal cancer (CRC) may be related to the presence of cancer stem cells (CSCs), but the underlying mechanism(s) remain unclear. Carcinoma-associated fibroblasts (CAFs) are intimately involved in tumor recurrence, and targeting them increases chemo-sensitivity. We investigated whether fibroblasts might increase CSCs thus mediating chemotherapy resistance. CSCs were isolated from either patient-derived xenografts or CRC cell lines based on expression of CD133. First, CSCs were found to be inherently resistant to cell death induced by chemotherapy. In addition, fibroblast-derived conditioned medium (CM) promoted percentage, clonogenicity and tumor growth of CSCs (i.e., CD133+ and TOP-GFP+) upon treatment with 5-fluorouracil (5-Fu) or oxaliplatin (OXA). Further investigations exhibited that exosomes, isolated from CM, similarly took the above effects. Inhibition of exosome secretion decreased the percentage, clonogenicity and tumor growth of CSCs. Altogether, our findings suggest that, besides targeting CSCs, new therapeutic strategies blocking CAFs secretion even before chemotherapy shall be developed to gain better clinical benefits in advanced CRCs.

Detection of Cyclin B1 Expression in G1-Phase Cancer Cell Lines and Cancer Tissues by Postsorting Western Blot Analysis

Protein complex of cyclin B1 and cyclin-dependent protein kinase 1 induces phosphorylation of key substrates that mediate cell cycle transition during the G2-M phase. It is believed that cyclin B1 accumulates in the S phase of the cell cycle and reaches the maximal level at mitosis but is absent in G1-phase cells. In the present study, we demonstrated that cyclin B1 was expressed in the arrested G1-phase MOLT-4 lymphocyte leukemia cells and in G1 phase T-7 transitional tumor cells, as determined by flow cytometry. In addition, we showed that cyclin B1 was detected in the G1 phase in breast cancer cells from patient tissues and in lymphocytes from patients with leukemia. These findings were confirmed for the first time by postsorting Western blot analysis and by confocal microscopy. Furthermore, by using postsorting Western blotting, we found that cyclin B1 was expressed in different time-window sections of the G1 phase under different conditions. For the asynchronously growing T-7 cells, cyclin B1 was detected in early G1 phase, whereas in MOLT-4 cells arrested in G1-S phase, cyclin B1 was mainly detected in late G1 phase. We propose that the cyclin B1 expressed in the G1 phase may differ from that expressed in the G2-M phase, and that this unscheduled type of cyclin B1 may play an important role in tumorigenesis and apoptosis.

New method for the analysis of Cell cycle-specific apoptosis

In this study, a new method for the analysis of cell cycle specificity of apoptosis was designed by using a modified annexin V and propidium iodide (API) method.

hSav1 interacts with HAX1 and attenuates its anti-apoptotic effects in MCF-7 breast cancer cells.

It has been reported that Salvador (SAV) is a core component of the Salvador-Warts-Hippo (SWH) pathway that restricts cell number, by functioning as a dual regulator of cell proliferation and apoptosis in Drosophila. However, the function of its human ortholog hSav1 (also called hWW45) in mammalian cells is poorly understood. In this study, we identified hematopoietic cell-specific protein 1 (HS1)-associated protein X-1 (HAX1), a 35-kDa protein localized to cell mitochondria, as a novel binding partner of hSav1 using a yeast two-hybrid screening technique. Our finding was confirmed by immunoprecipitation and glutathione-S-transferase (GST) pull-down assays of both proteins. Using immunofluorescence staining, we showed that HAX1 and hSav1 interact with each other. Analysis of the anti-apoptotic function of HAX1 revealed that the presence of hSav1 attenuated the HAX1 protective effects from hydrogen peroxide (H2O2)-induced cell death in MCF-7 cells, while knockdown of hSav1 by small interfering RNAs (siRNAs) significantly enhanced the anti-apoptotic function of HAX1. Also, using the Oncomine database, we found several studies in which HAX1 levels were significantly up-regulated and hSav1 expression was down-regulated in breast cancer samples compared to normal breast tissue. In summary, we conclude that hSav1 interacts with HAX1 and attenuates its protective role against apoptosis in MCF-7 breast cancer cells.

Role of RANTES and its receptor in gastric cancer metastasis.

This study examined the role of regulated upon activation normal T cell expressed and secreted (RANTES) and its receptor C-C chemokine receptor type 5 (CCR5) in gastric cancer metastasis and the associated mechanism. The expression of RANTES and CCR5 was detected by using immunohistochemical staining and Western blotting in the gastric cancer tissues obtained from 60 gastric cancer patients with or without lymph node metastasis (n=30 in each). The results showed that the expression levels of RANTES and CCR5 were higher in gastric cancer with lymph node metastasis than in that without metastasis (P<0.05). The expression levels of RANTES in 30 lymph nodes with cancerous invasion were higher than in 30 normal lymph nodes (P<0.05). Chemotactic test revealed that the number of migrating gastric cancer cells (n=295.0±54.6) induced by the protein of cancer-invading lymph nodes was greater than that by the protein mixture from cancer-invading lymph nodes and RANTES antibody (n=42.5±11.6) (P<0.05). RT-PCR showed that the expression levels of the main Th1 cytokines (IL-2, Γ-IFN) were lower in gastric cancer with lymph node metastasis (2.22±0.90, 3.26±1.15 respectively) than in that without metastasis (3.07±1.67, 4.77±1.52 respectively) (P<0.05), but the expression level of the main Th 2 cytokine (IL-10) was higher in gastric cancer with lymph nodes metastasis (6.06±2.04) than in that without metastasis (4.88±1.87) (P<0.05). It was concluded that RANTES and its receptor CCR5 may contribute to gastric cancer metastasis through influencing the balance of Th1/Th2. RANTES and CCR5 may become a marker of gastric cancer metastasis.SummaryThis study examined the role of regulated upon activation normal T cell expressed and secreted (RANTES) and its receptor C-C chemokine receptor type 5 (CCR5) in gastric cancer metastasis and the associated mechanism. The expression of RANTES and CCR5 was detected by using immunohistochemical staining and Western blotting in the gastric cancer tissues obtained from 60 gastric cancer patients with or without lymph node metastasis (n=30 in each). The results showed that the expression levels of RANTES and CCR5 were higher in gastric cancer with lymph node metastasis than in that without metastasis (P<0.05). The expression levels of RANTES in 30 lymph nodes with cancerous invasion were higher than in 30 normal lymph nodes (P<0.05). Chemotactic test revealed that the number of migrating gastric cancer cells (n=295.0±54.6) induced by the protein of cancer-invading lymph nodes was greater than that by the protein mixture from cancer-invading lymph nodes and RANTES antibody (n=42.5±11.6) (P<0.05). RT-PCR showed that the expression levels of the main Th1 cytokines (IL-2, Γ-IFN) were lower in gastric cancer with lymph node metastasis (2.22±0.90, 3.26±1.15 respectively) than in that without metastasis (3.07±1.67, 4.77±1.52 respectively) (P<0.05), but the expression level of the main Th 2 cytokine (IL-10) was higher in gastric cancer with lymph nodes metastasis (6.06±2.04) than in that without metastasis (4.88±1.87) (P<0.05). It was concluded that RANTES and its receptor CCR5 may contribute to gastric cancer metastasis through influencing the balance of Th1/Th2. RANTES and CCR5 may become a marker of gastric cancer metastasis.

Absence of DAB2IP promotes cancer stem cell like signatures and indicates poor survival outcome in colorectal cancer

Metastasis is a critical factor for the high mortality of colorectal cancer (CRC), but its mechanism is not completely understood. Epithelial-mesenchymal transition (EMT) is thought to play a key role in metastasis and also increases the cancer stem cell (CSC) feature that facilitates metastatic colonization. In this study, we investigated the biological roles of DAB2IP regulating EMT and stem cell–like features in human CRC. We demonstrate that DAB2IP suppresses NF-κB-mediated EMT and CSC features in CRC cells. In DAB2IP knockout mice, we discovered the hyperplasia in colonic epithelium which aberrantly represents the mesenchymal feature and NF-κB pathway activation. In clinic CRC tissue, we also reveal that reduced DAB2IP can enrich the CD133+ subpopulation. DAB2IP expression was inversely correlated with tumor differentiation and metastasis, and patients with lower DAB2IP expression had shorter overall survival time. Taken together, our study demonstrates that DAB2IP inhibits NF-κB-inducing EMT and CSC to suppress the CRC progression, and also suggests that DAB2IP is a beneficial prediction factor for CRC patient prognosis.

Are progenitor cells pre‐programmed for sequential cell cycles not requiring cyclins D and E and activation of Cdk2?

Abstract.  Objectives: Based on studies of unicellular organisms or cultured mammalian cells, the generally accepted model of cell‐cycle regulation has been developed in which sequential (scheduled) expression of cyclins D, E, A and B and activation of Cdk2 and Cdk1 takes place. It is assumed that the same model is applicable both in vivo and in vitro. Materials and methods: In the present study, we compared proliferating marrow cells freshly isolated from healthy individuals with proliferating lymphocytes in cultures. Results: We demonstrate that during progression of freshly collected human bone marrow cells through G1, S and G2/M, only Cdk1 combined with cyclins A and B1 was distinctly present and active, and its activity gradually increased. In contrast, in vitro growing mitogen‐stimulated lymphocytes had perfectly scheduled sequential expression of all four cyclins and Cdk1 and Cdk2 activities. Conclusion: Our findings demonstrate that the pattern of cyclin expression and Cdk activity in bone marrow in vivo is distinctly different from the one observed for normal cells in vitro. Because proliferating bone marrow cells are predominantly expanding populations of committed progenitors, it is likely that during the expansion phase their cell‐cycle progression is pre‐programmed, being driven solely by Cdk1 combined either with cyclin A or with cyclin B1. Expansion of progenitor cells thus may not require the early steps of cell‐cycle regulation, associated with triggering progression by availability of growth factors and mitogens.

KDM4B plays an important role in mitochondrial apoptosis by upregulating HAX1 expression in colorectal cancer

Histone methyltransferases and demethylases regulate transcription by altering the epigenetic marks on histones in tumorigenesis. Members of the histone lysine(K)-specific demethylase 4 (KDM4) family are dysregulated in several types of cancer. Here, we report a novel role for KDM4B in mitochondrial apoptosis. In this study, we demonstrate that KDM4B is overexpressed in colorectal cancer (CRC) tissues. Decreased expression of KDM4B significantly promoted apoptosis of CRC cell lines. Moreover, our data indicate that HAX1 is required for KDM4B-mediated mitochondrial apoptosis. The transcription of HAX1 was directly activated by KDM4B. We also show that HAX1 is overexpressed in CRC tissues and is positively correlated with KMD4B expression. Collectively, we demonstrate that KDM4B may play an important role in mitochondrial apoptosis and represent a potential therapeutic cancer target in CRC.

Expression of HAX-1 in colorectal cancer and its role in cancer cell growth

Colorectal cancer (CRC) is one of the most common types of cancer worldwide. Hematopoietic cell-specific protein 1-associated protein X-1 (HAX-1) has been found to be involved in several types of cancer. However, the role of HAX-1 in CRC remains to be elucidated. The aim of the present study was to investigate whether the expression of HAX-1 is associated with the progression of CRC, and to determine the effects of HAX-1 on the apoptosis and proliferation of CRC cells. Tumor tissues and adjacent noncancerous tissues were collected from 60 patients with CRC, following the provision of informed consent. The expression levels of HAX-1 and the association with clinical and pathological characteristics were then analyzed. The expression levels of HAX-1 were significantly higher in the cancerous tissues from the patients with CRC, particularly in tissues of an advanced stage of cancer. In addition, HAX-1 expression was associated with malignant progression and poor prognosis. Furthermore, SW480 CRC cells, overexpressing HAX-1, exhibited increased resistance to camptothecin in vitro, and promoted proliferation in vitro and in vivo. By contrast, HAX-1 knockdown significantly decreased the proliferation. In addition, the expression levels of ki-67 and phosphorylatedakt were inhibited following HAX-1 knockdown. In conclusion, the expression levels of HAX-1 were increased in cancerous tissue from patients with CRC, and were associated with progression of the disease. These results suggested that HAX-1 may contribute to chemotherapy resistance and malignant progression in CRC.

DNA damage response in resting and proliferating peripheral blood lymphocytes treated by camptothecin or X-ray.

DNA damage response (DDR) in different cell cycle status of human peripheral blood lymphocytes (PBLs) and the role of H2AX in DDR were investigated. The PBLs were stimulated into cell cycle with phytohemagglutinin (PHA). The apoptotic ratio and the phosphorylation H2AX (S139) were flow cytometrically measured in resting and proliferating PBLs after treatment with camptothecin (CPT) or X-ray. The expressions of γH2AX, Bcl-2, caspase-3 and caspase-9 were detected by Western blotting. DDR in 293T cells was detected after H2AX was silenced by RNAi method. Our results showed that DNA double strand breaks (DSBs) were both induced in quiescent and proliferating PBLs after CPT or X-ray treatment. The phosphorylation of H2AX and apoptosis were more sensitive in proliferating PBLs compared with quiescent lymphocytes (P<0.05). The expression levels of anti-apoptotic proteins Bcl-2 were reduced and cleaved caspase-3 and caspase-9 were increased. No significant changes were observed in CPT-induced apoptosis in 293T cells between H2AX knocking down group and controls. It was concluded that proliferating PBLs were more vulnerable to DNA damage compared to non-stimulated lymphocytes and had higher apoptosis rates. γH2AX may only serve as a marker of DNA damage but exert no effect on apoptosis regulation.SummaryDNA damage response (DDR) in different cell cycle status of human peripheral blood lymphocytes (PBLs) and the role of H2AX in DDR were investigated. The PBLs were stimulated into cell cycle with phytohemagglutinin (PHA). The apoptotic ratio and the phosphorylation H2AX (S139) were flow cytometrically measured in resting and proliferating PBLs after treatment with camptothecin (CPT) or X-ray. The expressions of γH2AX, Bcl-2, caspase-3 and caspase-9 were detected by Western blotting. DDR in 293T cells was detected after H2AX was silenced by RNAi method. Our results showed that DNA double strand breaks (DSBs) were both induced in quiescent and proliferating PBLs after CPT or X-ray treatment. The phosphorylation of H2AX and apoptosis were more sensitive in proliferating PBLs compared with quiescent lymphocytes (P<0.05). The expression levels of anti-apoptotic proteins Bcl-2 were reduced and cleaved caspase-3 and caspase-9 were increased. No significant changes were observed in CPT-induced apoptosis in 293T cells between H2AX knocking down group and controls. It was concluded that proliferating PBLs were more vulnerable to DNA damage compared to non-stimulated lymphocytes and had higher apoptosis rates. γH2AX may only serve as a marker of DNA damage but exert no effect on apoptosis regulation.