[Infiltrated T Cells Induce the Accumulation of Myeloid-derived Suppressor Cells in Tumor Microenvironment].

OBJECTIVE: To determine the role of infiltrated T cells in the accumulation of myeloid-derived suppressor cells (MDSCs) in tumor microenvironment. METHODS: T-cell-deficient nude mice models were established using BALB/c mice. Growth of tumors was compared between those with and without adoptive transfer of T cells. Pathological changes of the tumors were examined with HE histological analysis. The levels of MDSCs were detected with flow cytometry (FACS). RESULTS: Tumor growth was promoted in T-cell-deficient nude mice, which was accompanied with lower levels of MDSCs compared with BALB/c mice (P < 0.05). T cell transfer increased the level of MDSCs significantly (P < 0.05). T cells depletion decreased the level of MDSCs (P < 0.05). CONCLUSION: Infiltrated T cells induce the accumulation of MDSCs in tumor microenvironment, and influence tumor growth.

Pemetrexed induces G1 phase arrest and apoptosis through inhibiting Akt activation in human non small lung cancer cell line A549.

Pemetrexed is an antifolate agent which has been used for treating malignant pleural mesothelioma and non small lung cancer in the clinic as a chemotherapeutic agent. In this study, pemetrexed inhibited cell growth and induced G1 phase arrest in the A549 cell line. To explore the molecular mechanisms of pemetrexed involved in cell growth, we used a two-dimensional polyacrylamide gel electrophoresis (2-DE) proteomics approach to analyze proteins changed in A549 cells treated with pemetrexed. As a result, twenty differentially expressed proteins were identified by ESI-Q-TOF MS/MS analysis in A549 cells incubated with pemetrexed compared with non-treated A549 cells. Three key proteins (GAPDH, HSPB1 and EIF4E) changed in pemetrexed treated A549 cells were validated by Western blotting. Accumulation of GAPDH and decrease of HSPB1 and EIF4E which induce apoptosis through inhibiting phosphorylation of Akt were noted. Expression of p-Akt in A549 cells treated with pemetrexed was reduced. Thus, pemetrexed induced apoptosis in A549 cells through inhibiting the Akt pathway.

RNA interference of Biot2 induces G1 phase arrest and apoptosis in mouse colorectal cancer cell line.

Biot2 is a tumor-associated antigen, and it is a novel gene (GenBank EF100607) that was first identified with the SEREX technique and named by our laboratory. It is highly expressed in cancer cells and testis, with low or no expression in normal tissues. In our previous study, RNA interference of human Biot2 can inhibit tumor cell growth, and it is associated with poor prognosis of patients in clinical study; however, the mechanism of Biot2 that effects tumor growth is not yet clear. Here, in this study, we explore further the mechanism of Biot2 by silencing Biot2 in CT26 cells. It provides some theoretical basis for Biot2 as a new target for gene therapy. In CT26 cells, the expression of Biot2 was downregulated by Biot2-shRNA. It also promoted G1 phase arrest, the expression of p16 and p21, and cell apoptosis. In the mouse model, the tumor volume and the expression of PCNA of the Biot2-shRNA group significantly decreased. These results suggest that silencing Biot2 in CT26 cells by RNA interference can inhibit cell growth in vitro and in vivo. It also induces cell cycle arrest in the G1 phase and apoptosis throughout regulation of p16 and p21. Taken together, our data demonstrate that Biot2 can be a potential target of gene therapy.

Treatment of dextran sodium sulfate-induced experimental colitis by adoptive transfer of peritoneal cells.

The adoptive transfer of the natural regulatory B cells and macrophages should be a useful treatment for inflammation and autoimmune disease. However, it is usually difficult to isolate these cells from the tissues and expand them. Here, we investigated the feasibility of adoptively transferring peritoneal cells (PCs) as a treatment for DSS-induced colitis. We found that peritoneal cavity can provide an easily accessible site for harvesting enough number of PCs, namely, two-dose PCs for the treatment from a mouse in one operation. Adoptive therapy of these cells from healthy mice or those with disease is effectively in reducing the disease activity score. The natural B cells and macrophages of the infused PCs can selectively migrate to lesion sites and regulate the expression of Stat3, NF-κB, Smad3 and Smad7. Additionally, PCs exert dual activity of IL-10 and TGF-ß secreted spontaneously by both peritoneal B cells and macrophages, which in turn enhance the induction of regulatory B cells and Macrophages in microenvironment of inflammation. Moreover, PCs can re-establish immunological tolerance in the OVA-immunized mice. Thus, our findings provide a new strategy for colitis therapy and could be of importance in additional exploration of other inflammation and autoimmune diseases therapy.

Phosphorylation and changes in the distribution of nucleolin promote tumor metastasis via the PI3K/Akt pathway in colorectal carcinoma.

Here, we investigated the molecular mechanism underlying the changes in the distribution of nucleolin. Our study identified PI3K/Akt signaling as an essential pathway regulating the distribution of nucleolin. Furthermore, nucleolin can interact with phospho-PI3K-p55, and changes in the distribution of nucleolin were related to its phosphorylation. Subsequently, we analyzed the correlation of VEGF and nucleolin, and found that distribution of nucleolin related to metastatic potential. Finally, blocking cell surface nucleolin influences the process of epithelial-mesenchymal transitions. This indicates that nucleolin may be a novel cancer therapy target and a predictive marker for tumor migration in colorectal carcinoma.

Synergic antitumor effect of SKLB1002 and local hyperthermia in 4T1 and CT26.

A de novo VEGFR2-inhibited compound SKLB1002 which is independently developed in our laboratory has been described for antiangiogenesis and displays a potent antitumor activity in vivo and in vitro. In the present investigation, we aim to prove that combination therapy of SKLB1002 with hyperthermia plays a synergy as an antitumor agent in solid tumor. In this study, we analyzed their synergetic inhibitory action on human umbilical vein endothelial cells (HUVEC), murine mammary cancer 4T1, murine colon carcinoma CT26 in vitro. Multiply-table tournament was performed to detect cell proliferation in vitro. 4T1 implantation and CT26 implantation in BALB/c mice were used to examine the activity of combination therapy of SKLB1002 with hyperthermia in vivo. Vascular density was determined by CD31 immunohistochemistry. TUNEL was used to measure apoptosis in tumor tissue. Metastasis assay was investigated via measurement of pulmonary metastasis nodules under the microscope. Potential toxicity of combination therapy was observed by histologic analysis of main organs stained with H&E. In vitro, the combination therapy significantly inhibited cell proliferation of HUVEC, 4T1 and CT26. In vivo, 4T1 and CT26 model experiments showed that combination therapy remarkably inhibited tumor growth and prolonged life span. When compared with controls, combination therapy reached 61 % inhibition index of tumor growth against CT26 and 51 % against 4T1. Moreover, it reduced angiogenesis and increased tumor apoptosis and necrosis. It was further found that combination therapy could efficiently prevent tumor from metastasizing to lung. Importantly, it had no toxicity to main organs including heart, liver, spleen, lung and kidney. Combination treatment has been proved to be a novel and strong strategy in clinical antitumor therapy. Our findings suggest that the combination therapy of SKLB1002 with hyperthermia has a synergistic antiangiogenesis, anticancer and promotion of apoptosis efficacy compared with controls. These findings could pave a new way in clinical tumor therapy.

Inhibition of lymphatic metastases by a survivin dominant-negative mutant.

Metastasis is the most lethal attribute of human malignancy. High-level expression of survivin is involved in both carcinogenesis and angiogenesis in cancer. Previous studies indicate that a mutation of the threonine residue at position 34 (Thr34Ala) of survivin generates a dominant-negative mutant that induces apoptosis, inhibits angiogenesis, and suppresses highly metastatic breast carcinoma in mouse models. We investigated the efficacy of gene therapy with a survivin dominant-negative mutant and possible factors related to lymph node metastasis. The metastasis rate was compared between each group in order to find a survivin-targeted therapy against lymphangiogenesis in its earliest stages. We established lymph node metastasis models and treated animals with H22 tumors with Lip-mSurvivinT34A (Lip-mS), Lip-plasmid (Lip-P), or normal saline (NS). Eight days after the last dose, five randomly chosen mice from each group were sacrificed. We detected the apoptotic index, microvessel density (MVD), lymphatic microvessel density (LMVD), and the expression of VEGF-D with immunohistochemistry. After the remaining animals were sacrificed, we compared the tumor-infiltrated lymph nodes in each group. Administration of mSurvivinT34A plasmid complexed with cationic liposome (DOTAP/chol) resulted in the efficacious inhibition of tumor growth and lymph node metastasis within the mouse H22 tumor model. These responses were associated with tumor cell apoptosis, and angiogenesis and lymphangiogenesis inhibition. Our results suggested that Lip-mSurvivinT34A induced apoptosis and inhibited tumor angiogenesis and lymphangiogenesis, thus suppressing tumor growth and lymphatic metastasis. The mSurvivinT34A survivin mutant is a promising strategy of gene therapy to inhibit lymphatic metastasis.

[Clinical observation on meridian detection and diagnosis in 66 patients with hypertension].

OBJECTIVE: To observe the meridian reaction in patients with hypertension, to explore the significance of meridian diagnosis. METHODS: Sixty-six patients with hypertension received two different methods of meridian detection. Kang Wei Human Body Meridian Analysis and Diagnosis System was applied to detect the volt-ampere characteristic curve on the Yuan (source) point of the 12 meridians. Five minutes after the first examination, the traditional method of meridian detection was used to examine the meridian reactions, such as pressing pain, tuber and cord-like reactants along the 12 meridians at forearms, lower limb, head and back. RESULTS: There were sixty-five cases of abnormal volt-ampere characteristic curve among sixty-six patients with hypertension. The number of cases about Kidney Meridian of Foot-Shaoyin and Bladder Meridian of Foot-Taiyang were both 28 with the highest abnormal rate being 43.1%; Using traditional method of meridian detection, there were 41 cases of abnormal meridian reactions, about 28 cases of Governor Vessel, with the highest abnormal rate being 68.3%. CONCLUSION: The abnormal meridian reactions exist in Governor Vessel, Kidney Meridian and Bladder Meridian in patients with hypertension. Therefore, this finding provides clinical evidence in meridian diagnosis.