Xinxin Bu

Effects of Inflammatory Factors on Mesenchymal Stem Cells and Their Role in the Promotion of Tumor Angiogenesis in Colon Cancer

Mesenchymal stem cells (MSCs), which are modulated by cytokines present in the tumor microenvironment, play an important role in tumor progression. It is well documented that inflammation is an important part of the tumor microenvironment, so we investigated whether stimulation of MSCs by inflammatory cytokines would contribute to their ability to promote tumor growth. We first showed that MSCs could increase C26 colon cancer growth in mice. This growth-promoting effect was further accelerated when the MSCs were pre-stimulated by inflammatory factors IFN-γ and TNF-α. At the same time, we demonstrated that MSCs pre-stimulated by both inflammatory factors could promote tumor angiogenesis in vivo to a greater degree than untreated MSCs or MSCs pre-stimulated by either IFN-γ or TNF-α alone. A hen egg test-chorioallantoic membrane (HET-CAM) assay showed that treatment of MSC-conditioned medium can promote chorioallantoic membrane angiogenesis in vitro, especially treatment with conditioned medium of MSCs pretreated with IFN-γ and TNF-α together. This mechanism of promoting angiogenesis appears to take place via an increase in the expression of vascular endothelial growth factor (VEGF), which itself takes place through an increase in signaling in the hypoxia-inducible factor 1α (HIF-1α)-dependent pathway. Inhibition of HIF-1α in MSCs by siRNA was found to effectively reduce the ability of MSC to affect the growth of colon cancer in vivo in the inflammatory microenviroment. These results indicate that MSCs stimulated by inflammatory cytokines such as IFN-γ and TNF-α in the tumor microenvironment express higher levels of VEGF via the HIF-1α signaling pathway and that these MSCs then enhance tumor angiogenesis, finally leading to colon cancer growth in mice.

Increased p38-MAPK is responsible for chemotherapy resistance in human gastric cancer cells.

BackgroundChemoresistance is one of the main obstacles to successful cancer therapy and is frequently associated with Multidrug resistance (MDR). Many different mechanisms have been suggested to explain the development of an MDR phenotype in cancer cells. One of the most studied mechanisms is the overexpression of P-glycoprotein (P-gp), which is a product of the MDR1 gene. Tumor cells often acquire the drug-resistance phenotype due to upregulation of the MDR1 gene. Overexpression of MDR1 gene has often been reported in primary gastric adenocarcinoma.MethodsThis study investigated the role of p38-MAPK signal pathway in vincristine-resistant SGC7901/VCR cells. P-gp and MDR1 RNA were detected by Western blot analysis and RT-PCR amplification. Mitgen-activated protein kinases and function of P-gp were demonstrated by Western blot and FACS Aria cytometer analysis. Ap-1 activity and cell apoptosis were detected by Dual-Luciferase Reporter Assay and annexin V-PI dual staining.ResultsThe vincristine-resistant SGC7901/VCR cells with increased expression of the multidrug-resistance 1 (MDR1) gene were resistant to P-gp-related drug and P-gp-unrelated drugs. Constitutive increases of phosphorylated p38-MAPK and AP-1 activities were also found in the drug-resistant cells. Inhibition of p38-MAPK by SB202190 reduced activator protein-1 (AP-1) activity and MDR1 expression levels and increased the sensitivity of SGC7901/VCR cells to chemotherapy.ConclusionActivation of the p38-MAPK pathway might be responsible for the modulation of P-glycoprotein-mediated and P-glycoprotein-unmediated multidrug resistance in the SGC7901/VCR cell line.

Autophagic cell death induced by 5-FU in Bax or PUMA deficient human colon cancer cell

Autophagy is a membrane process that results in the transporting of cellular contents to lysosomes for degradation. Autophagic cell death is another way of programed cell death called type II PCD, which has complicated connection with apoptosis, both of these two types of cell death play an important role in tumor development. In this study, we investigated chemotherapeutic agent induced cell death pathway in wild type (WT), Bax(-/-) and PUMA(-/-) HCT116 cells. Bax or PUMA deficient cells had similar chemosensitivity to WT cells but were defective in undergoing apoptosis. The results of electron microscopy and GFP-LC3 localization assay showed that autophagy was induced in Bax or PUMA deficient cells but not in WT cells. mTOR activity was decreased in Bax or PUMA deficient cells which further indicated the up-regulation of autophagy. Inhibition of autophagy by 3-Methyladenine (3-MA) decreased the cell death in Bax or PUMA deficient cells. Taken together, these results suggest that autophagic cell death can be used as an alternative cell death pathway in apoptosis defective cells and may bring a new target for cancer therapy.

Immunosuppressive effect of bone marrow-derived mesenchymal stem cells in inflammatory microenvironment favours the growth of B16 melanoma cells.

Mesenchymal stem cells (MSCs) are studied for their potential clinical use in regenerative medicine, tissue engineering and tumour therapy. However, the therapeutic application of MSCs in tumour therapy still remains limited unless the immunosuppressive role of MSCs for tumour growth in vivo is better understood. In this study, we investigated the mechanism of MSCs favouring tumour escape from immunologic surveillance in inflammatory microenvironment. We first compared the promotive capacity of bone marrow‐derived MSCs on B16 melanoma cells growth in vivo, pre‐incubated or not with the inflammatory cytokines interferon (IFN)‐γ and tumour necrosis factor (TNF)‐α. We showed that the development of B16 melanoma cells is faster when co‐injected with MSCs pre‐incubated with IFN‐γ and TNF‐α compared with control groups. Moreover, tumour incidence increases obviously in allogeneic recipients when B16 melanoma cells were co‐injected with MSCs pre‐incubated with IFN‐γ and TNF‐α. We then demonstrated that the immunosuppressive function of MSCs was elicited by IFN‐γ and TNF‐α. These cytokine combinations provoke the expression of inducible nitric oxide synthase (iNOS) by MSCs. The impulsive effect of MSCs treated with inflammatory cytokines on B16 melanoma cells in vivo can be reversed by inhibitor or short interfering RNA of iNOS. Our results suggest that the MSCs in tumour inflammatory microenvironment may be elicited of immunosuppressive function, which will help tumour to escape from the immunity surveillance.

Synergistic effect of mTOR inhibitor rapamycin and fluorouracil in inducing apoptosis and cell senescence in hepatocarcinoma cells.

Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide with an annual occurrence of one million new cases. At present there is no effective treatment for HCC individuals that not amenable to curative therapies. Recent studies show the PI3K/Akt/mTOR signal pathway is involved in multiple cellular functions including proliferation, differentiation, tumorigenesis, and apoptosis. Rapamycin (a specific mTOR inhibitor) could lead to G1 arrest of many malignant cell lines, and currently analogs of rapamycin are being investigated as a cancer chemotherapeutic adjuvant. This study investigated rapamycin and chemotherapeutic agent 5-fluorouracil (5-Fu) in combination treatment induced apoptosis and cell senescence in hepatocarcinoma cell line SMMC-7721 cells. Treating SMMC-7721 cells with rapamycin plus 5-Fu led to not only apoptosis but also cell senescence, and the senescent cells exhibited significantly less clonogenic potential than 5-Fu individually treated cells. Further study showed rapamycin plus 5-Fu-induced senescence-like growth arrest was accompanied by down-regulation of AP-1 and NF-κB transcription activity. These results suggest that inhibitors of mTOR may have anticancer potential when used together with some other chemotherapeutic agents, and that down-regulation of AP-1 and NF-κB transcription activity might take part in a senescence-like growth arrest program induced by rapamycin plus 5-Fu.

Correlation of CpG island methylator phenotype with poor prognosis in hepatocellular carcinoma

CpG island methylator phenotype (CIMP), in which multiple genes are concurrently methylated, is an important mechanism in hepatocellular carcinoma development. We determined a hypermethylation profile in hepatocellular carcinoma (HCC). We examined the promoter methylation status of 10 genes in 60 cases of hepatocellular carcinoma (HCC), 60 cases of paired non-tumor tissues, and 6 cases of normal tissues by methylation-specific PCR. The average methylated gene numbers were significantly different between HCC and nontumor tissues (p<0.001). We found metastasis, gamma-glutamyl transpeptidase (GGT) and tumor node metastasis (TNM) stage were significantly different among patients with different CIMP status. Patients with high frequency CIMP tumors had significantly worse survival than patients with intermediate frequency or no CIMP tumors (p<0.01 and p<0.05, respectively). Our results suggested that CIMP could serve as a molecular marker of late stage and poorly prognostic HCC development.

Coupled down-regulation of mTOR and telomerase activity during fluorouracil-induced apoptosis of hepatocarcinoma Cells

BackgroundHepatocellular carcinoma (HCC) is the most invasive and frequently diagnosed malignancy and the second leading cause of cancer death in many regions of Asia. The PI3K/Akt/mTOR signal pathway is involved in multiple cellular functions including proliferation, differentiation, tumorigenesis, and apoptosis. Up-regulation of telomerase activity is thought to be a critical step leading to cell transformation.MethodsThis study investigated changes in mTOR pathway and telomerase activity in hepatocarcinoma cell line SMMC-7721 treated with chemotherapeutic agent 5-fluorouracil (5-Fu). We detected apoptosis of hepatocarcinoma cells by TUNEL assay. Telomerase activity, hTERT transcription level and p- p70 S6k was demonstrated by the telomeric repeat amplification protocol and silver staining assay, Dual-Luciferase Reporter Assay and Western blot analysis respectively.ResultsTreating SMMC-7721 cells with 5-Fu leads to apoptosis of the cells, and reduction in telomerase activity, as well as a dramatic reduction in the activated form of p70 S6 kinase, a mTOR substrate. The 5-Fu treatment nearly abolishes transcription of hTERT (the major component of telomerase) mRNA. Treating SMMC-7721 cells with Rapamycin, a specific mTOR inhibitor, significantly reduce hTERT protein level but did not affect hTERT transcription. 5-Fu and rapamycin were synergistic in regards to down-regulation of telomerase activity in hepatocarcinoma cells.ConclusionThese results suggest that chemotherapeutic agent 5-Fu may down-regulate telomerase activity at both transcriptional level and PI3K/Akt/mTOR pathway-dependent post-transcriptional level to facilitate hepatocellular carcinoma cell apoptosis.

siRNA-mediated inhibition of hTERT enhances chemosensitivity of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide. However, there is no effective treatment for HCC. It has been shown that sustained activation of telomerase is essential for the growth and progression of HCC, suggesting that telomerase is a rational target for HCC therapy. Here, we investigated the effects of siRNA-mediated knockdown of hTERT, the catalytic and rate-limiting subunit of telomerase, on the sensitivity of HCC cells to Cisplatin. While silencing of hTERT and the resultant inhibition of telomerase activity by infection with the recombinant adenoviruse expressing a hTERT siRNA (Ad-si/hTERT) alone did not affect the proliferation and viability of SMMC7721 and HepG2 HCC cells within 5 days, co-administration of Ad-si/TERT, but not the empty adenovirus vector, with cisplatin caused much greater extent of apoptosis in vitro under the same conditions and induced significantly more robust inhibition of SMMC7721 and HepG2 tumors growth in a mouse tumor xenograft model than cisplatin monotherapy. Our results demonstrated the synergistic effect between hTERT siRNA and Cisplatin in the suppression of HCC progression and indicated that the combination of hTERT-specific siRNA and cisplatin could be an effective therapy for HCC.

Tumor necrosis factor-alpha promotes tumor growth by inducing vascular endothelial growth factor.

Tumor necrosis factor (TNF)-α has been proved as an adjuvant therapy for tumor by FDA. However, the effect of chronic TNF-α expression for tumor is still controversial. In this study, we investigated the effect of low-dose TNF-α on tumor growth. We confirmed that low-dose TNF-α promoted angiogenesis of tumor in vivo, vascular endothelial growth factor (VEGF) and hypoxia-inducible factor (HIF)-1α, the transcription factor of VEGF, were both upregulated. Our results suggested that low-dose TNF-α was a powerful activator of angiogenesis in tumor and HIF-1α-VEGF pathway seemed to be the most important molecular mechanism.

Clinical Significance of Telomerase Activity and Human Telomerase Reverse Transcriptase mRNA Expression for Differential Diagnosis of Malignant and Benign Liver Lesions

ObjectiveTo study the clinical significance of telomerase activity and human telomerase reverse transcriptase (hTERT) mRNA expression for differential diagnosis of malignant and benign liver lesions.MethodsTelomerase activity was determined by an ELISA -based telomeric repeat amplification protocol (ELISA-TRAP) assay on 130 surgical resected liver samples and 58 percutaneous biopsied liver samples. In addition, the samples were assayed for expression of hTERT mRNA measured by a reverse transcriptase-polymerase chain reaction (RT-PCR). Postoperative pathological examinations were also performed on these samples.ResultsAmong the 130 surgical liver samples, the positive rates of telomerase activity in hepatocellular carcinoma (HCC), liver cirrhosis and chronic hepatitis tissues were 85.9% (55/64), 25.0% (8/32) and 8.3% (2/24) respectively, and the positive rates of hTERT mRNA expression were 89.1% (57/64), 25.0% (8/32) and 8.3% (2/24) respectively. Neither telomerase activity nor hTERT mRNA expression was detected in 10 normal liver tissues. Among the 58 biopsied liver specimens, the positive rates of telomerase activity in HCC, cholangiocellular carcinoma, focal nodular hyperplasia, inflammatory pseudotumor and adenomatous hyperplasia tissues were 85.7% (30/35), 100% (4/4), 33.3% (4/12), 25.0% (1/4) and 33.3% (1/3) respectively, and the positive rates of hTERT mRNA expression were 88.6% (31/35), 100% (4/4), 33.3% (4/12), 25.0% (1/4) and 33.3% (1/3) respectively. The positive level of telomerase activity and hTERT mRNA expression in malignant liver tumors was significantly higher than that found in benign liver lesions (P< 0.01).ConclusionDetermination of telomerase activity or hTERT mRNA expression in percutaneous biopsied liver tissues may be useful for differential diagnosis in malignant and benign liver lesions.