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Understanding how the tumor microenvironment is shaped by various factors is important for the development of new therapeutic strategies. Tumor cells often undergo spontaneous apoptotic cell death in tumor microenvironment, these apoptotic cells are histologically co-localized with immunosuppressive macrophages. However, the mechanism by which tumor cell apoptosis modulates macrophage polarization is not fully understood. In this study, we aimed to explore the tumor promoting effects of apoptotic tumor cells and the signal pathways involved.Apoptotic cells and macrophages in tumors were detected by immunohistochemical staining. Morphological analysis was performed with Giemsa staining. Lipids generated from apoptotic cells were detected by liquid chromatography-mass spectrometry. Phosphatidylserine-containing liposomes were prepared to mimic apoptotic cells. The expression of protein was determined by real-time PCR, immunohistochemistry enzyme-linked immunosorbent assay and Western blotting. Mouse malignant ascites and subcutaneous tumor models were designed for in vivo analysis. Transgenic mice with specific genes knocked out and inhibitors specific to certain proteins were used for the mechanistic studies.The location and the number of apoptotic cells were correlated with that of macrophages in several types of carcinomas. Phosphatidylserine, a lipid molecule generated in apoptotic cells, induced polarization and accumulation of M2-like macrophages in vivo and in vitro. Moreover, sustained administration of phosphoserine promoted tumor growth in the malignant ascites and subcutaneous tumor models. Further analyses suggested that phosphoserine induced a M2-like phenotype in macrophages, which was related to the activation of phosphoserine receptors including T-cell immunoglobin mucin 4 (TIM4) and the FAK-SRC-STAT3 signaling pathway as well as elevated the expression of the histone demethylase Jumonji domain-containing protein 3 (JMJD3). Administration of specific inhibitors of these pathways could reduce tumor progression.This study suggest that apoptotic cell-generated phosphoserine might be a notable signal for immunosuppressive macrophages in tumors, and the related pathways might be potential therapeutic targets for cancer therapy.

Colorectal carcinoma (CRC) is the one of the most common cancers with considerable metastatic potential, explaining the need for new drug candidates that inhibit tumor metastasis. The signal transducers and activators of the transcription 3 (Stat3) signaling pathway has an important role in CRC and has been validated as a promising anticancer target for CRC therapy. In the present study, we report our findings on nifuroxazide, an antidiarrheal agent identified as an inhibitor of Stat3. Our studies showed that nifuroxazide decreased the viability of three CRC cell lines and induced apoptosis of cancer cells in a concentration-dependent manner. Moreover, western blot analysis demonstrated that the occurrence of its apoptosis was correlated with the activation of Bax and cleaved caspase-3, and decreased the expression of Bcl-2. In addition, nifuroxazide markedly impaired CRC cell migration and invasion by downregulating phosphorylated-Stat3

Doxorubicin (DOX) is a wide spectrum antitumor drug, but its clinical application is limited by the cardiotoxicity. Ghrelin, a multi-functional peptide hormone with metabolic regulation in energy homeostasis, plays important roles in cardiovascular protection. Now, the underlying mechanisms of ghrelin against DOX-induced cardiomyocyte apoptosis and atrophy are still not clear. In the present study, we revealed an autophagy-dependent mechanism involved in ghrelin's protection against DOX-induced cardiomyocyte death and size decrease. We observed that DOX insult induced remarkable mortality and cardiac dysfunction in mice, and increase in LDH leakage, cardiomyocyte apoptosis and decrease in cell viability and size in mouse hearts and H9c2 cell cultures, which were effectively improved by ghrelin supplement. We further observed that the strong autophagy stirred by DOX exposure was paralleling with the serious apoptosis and size decrease in cardiomyocytes. Ghrelin, like an autophagy inhibitor, 3-MA, inhibited the DOX-induced autophagy and attenuated cardiomyocyte apoptosis and size decrease. Furthermore, ghrelin significantly reduced the intercellular oxidative stress level, a strong autophagy trigger, partly by augmenting the expression and activities of the endogenous anti-oxidative enzymes. After the further investigation in the post signaling pathways of ghrelin receptors in H9c2 cells, including ERK, p38/MAPK, JNK, AMPK and Akt, we observed that ghrelin supplement only reduced the DOX-activated AMPK and augmented the DOX-down regulated p38-MAPK and mTOR phosphorylation. Our results indicated that ghrelin effectively improved the cardiomyocyte survival and size maintenance by suppressing the excessive autophagy through both ROS inhibition and mTOR induction through suppressing AMPK activity and stimulating p38-MAPK activity.

d These authors contributed equally to this work Abstract Background: AZD4547, a small-molecule inhibitor targeting the tyrosine kinase of Fibroblast Growth Factor Receptors (FGFRs), is currently under phase II clinical study for human subjects having breast cancer, while the underlying mechanism remains elusive. The aim of this study is to explore the potential mechanism by which AZD4547 inhibits breast tumor lung metastases at the level of the tumor microenvironment. Methods: First, through in vitro experiments, we investigated the efficacy of the FGFRs inhibitor AZD4547 on 4T1 tumor cells for their proliferation, apoptosis, migration, and invasion. Second, by in vivo animal experiments, we evaluated the effects of AZD4547 on tumor growth and lung metastases in 4T1 tumor-bearing mice. Finally, we examined the impact of AZD4547 on the infiltration of myeloid-derived suppressor cells (MDSCs) in lung, spleens, peripheral blood and tumor. Results: Through this study we found that AZD4547 could efficiently suppress tumor 4T1 cells through restraining their proliferation, blocking migration and invasion, and inducing apoptosis in vitro. In animal model we also demonstrated that AZD4547 was able to inhibit tumor growth and lung metastases, consistent with the decreased MDSCs accumulation in the tumor and lung tissues, respectively. Moreover, the reduced number of MDSCs in peripheral blood and spleens were also observed in the AZD4547-treated mice. Importantly, through the AZD4547 treatment, the CD4 + and CD8 + T-cells were significantly increased in tumor and spleens. Conclusion: Our studies showed that AZD4547 can inhibit breast cancer cell proliferation, induce its apoptosis and block migration and invasion in vitro and suppress tumor growth and lung metastases by modulating the tumor immunologic microenvironment in vivo.

Transfection of plasmid vectors coexpressing short hairpin RNA (shRNA) for focal adhesion kinase (FAK) and epidermal growth factor receptor (EGFR) significantly inhibited protein level of FAK and EGFR. Knockdown of FAK and EGFR expression significantly inhibited cell proliferation and induced cell apoptosis of A549 lung cancer cells in vitro. In A549 subcutaneous xenograft model, mice treated for 3 weeks with plasmid that coexpresses FAK and EGFR shRNA had significantly smaller tumors than those in control mice (P

Fibroblast activation protein (FAP) is a specific serine protease expressed in tumor stroma proven to be a stimulatory factor in the progression of some cancers. The purpose of this study was to investigate the effects of FAP knockdown on tumor growth and the tumor microenvironment. Mice bearing 4T1 subcutaneous tumors were treated with liposome-shRNA complexes targeting FAP. Tumor volumes and weights were monitored, and FAP, collagen, microvessel density (MVD), and apoptosis were measured. Our studies showed that shRNA targeting of FAP in murine breast cancer reduces FAP expression, inhibits tumor growth, promotes collagen accumulation (38%), and suppresses angiogenesis (71.7%), as well as promoting apoptosis (by threefold). We suggest that FAP plays a role in tumor growth and in altering the tumor microenvironment. Targeting FAP may therefore represent a supplementary therapy for breast cancer. [BMB Reports 2013; 46(5): 252-257]

Heme oxygenase-1 [HO-1, also called heat shot protein 32 (HSP32)] can specifically metabolize heme to carbon monoxide, biliverdin, and ferrous iron and plays an important role in the processes of anti-inflammation, tissue protection, and antioxidative stress reaction. It has been reported that HO-1 can promote tumorigenesis and metastasis of many tumors. However, the detailed mechanisms of how HO-1 affects tumor progress are not clear. Here, we used ZnPPIX (a specific inhibitor of HO-1) to evaluate its potential effects on mouse breast cancer and tumor-associated macrophages (TAMs). We found out that mouse 4T1 breast cancer growth can be effectively suppressed through inhibition of HO-1 in vitro and in vivo. Moreover, in the 4T1 mouse model, when HO-1 was suppressed in TAMs, alternatively activated macrophages (M2 type) switched to classically activated macrophages (M1 type). In conclusion, 4T1 breast cancer growth was modulated by HO-1 expression. Furthermore, inhibition of HO-1 may induce tumor-associated immune response by activating TAMs' alternative proliferation. These data suggest that HO-1 may be an important target of breast cancer treatment.

Anti-angiogenesis has been a promising strategy for cancer therapy. However, many signal pathways are activated during anti-angiogenic treatment to counteract the therapeutic efficacy. Among these pathways, evidence has directly pointed to the phosphatidylinositol 3-kinase/Akt (PI3K/Akt) pathway, whose activation resulted in tolerance to the absence of nutrients and oxygen when tumor angiogenesis has been inhibited. In the present study, we investigated the effects of blocking activation of the PI3K/Akt pathway on cell survival in vitro and tumor growth in vivo during anti-angiogenesis therapy. In modeled microenvironments in vitro, we observed that the phosphorylation of Akt in tumor cells was increased gradually in the absence of serum and oxygen in a time-dependent manner. The specific inhibitors of PI3K inhibited the proliferation of tumor cells in a dose-dependent manner in vitro. Moreover, inhibition was enhanced gradually with increased serum deprivation and/or hypoxia. In a mouse tumor model, we found the phosphorylation of Akt obviously increased following anti-angiogenic therapy using plasmids encoding soluble vascular endothelial growth factor receptor-2, but significantly reduced after treatment with LY294002. Consequently, the combinational treatment exhibited better antitumor effects compared with single treatments, presenting larger necrosis-like areas, more apoptotic cells, less microvessel density and less phosphorylated Akt in tumors. These results suggest that blocking activation of the PI3K/Akt pathway during anti-angiogenesis therapy could enhance antitumor efficacy. Thus, targeting the PI3K/Akt pathway might be a promising strategy to reverse tumor resistance to anti-angiogenesis therapy. (Cancer Sci 2011; 102: 1469–1475)

Deltonin, a steroidal saponin, isolated from Dioscorea zingiberensis Wright (DZW), has shown high-cytotoxic activity in cancer cells. However, its mechanisms and in vivo anti-cancer effects remain unknown. In the present study, we evaluated the effects and explored the anti-tumor mechanisms of deltonin on a panel of colon cancer cell lines and in a mouse model of murine colon cancer C26. Deltonin had more cytotoxic effect on C26 cells than 5-fluorouracil had, promoting dramatic G2-M phase arrest and apoptosis in C26 cells in a concentration-dependent manner; oral administration of deltonin significantly inhibited the tumor growth and prolonged survival of the tumor bearing mice. The deltonin treatment caused a noticeable apoptosis in tumor tissue, which associated with increased levels of Bax, activated caspase-3, caspase-9, and cleaved poly (ADPribose) polymerase, decreased pro-caspase-8, pro-caspase-9, Bcl-2 expression levels and extracellular signal-regulated kinase-1/2 activity; and dose-dependently inhibit angiogenesis. In conclusion, the findings in this study demonstrated that deltonin is an effective natural agent for cancer therapy, which may be mediated, in part, by induction of apoptosis, as well as involve mitogen-activated protein kinase pathways, and inhibition of angiogenesis.

Background Cyclin B1 (CLB1) is necessary for mitotic initiation in mammalian cells and plays important roles in cancer development. Therefore, a potential strategy in cancer therapy is to suppress the activity of CLB1 by delivering antisense constructs of CLB1 into tumor cells. In previous CLB1 studies, antisense constructs with a short half life were often used and these constructs might not persistently inhibit CLB1. Methods We successfully created a recombinant plasmid encoding the full-length antisense cDNA of mouse cyclin B1 (AS-mCLB1) and transfected this construct to the murine Lewis lung carcinoma (LL/2) and CT-26 colon carcinoma (CT-26) cells. We isolated clones of LL/2 and CT-26 transfectants with stable expression of AS-mCLB1. Reverse transcriptional polymerase chain reaction (RT-PCR) and Western blot were applied to detect the expression of the mRNA and protein levels of CLB1. To further test the efficacy of this strategy in vivo, AS-mCLB1-expressing LL/2 and CT-26 transfectants were implanted into mice. Results We found the expression of the mRNA and protein levels of CLB1 decrease in these transfectants. The inhibition of CLB1 caused prominent G1 arrest, abnormal morphology, retarded cell growth and an increase in apoptosis. In AS-mCLB1-expressing LL/2 and CT-26 transfectants implanted mice, tumorigenicity was effectively suppressed compared with the controls. In addition, the expression of AS-mCLB1 also significantly increases the survival duration of implanted animals. Conclusion AS-mCLB1 is likely to be useful in future cancer therapy, which may be associated with its ability to down-regulate the expression of CLB1 and then induce G1 arrest and apoptosis in tumor cells.

To investigate nanoparticles’ antitumor effect, a globose 30~35nm Ce(IV) doped Titanium dioxide nanoparticles (CDT) were prepared by impregnation method and characterized by X-ray diffraction (XRD) and transmission electron microscopy(TEM). Proliferation of BEL7402 human Hepatoma cells was studied in vitro by using fluorescence microscopy and Flow Cytometry. The results show that cerium elements doping enhanced thermal stability of nano-size titanium dioxide to 800°C. With UV irradiated for 8min, BEL7402 were induced a dose-dependent apoptosis by CDT, about 28.2% , 41.5% and 88.3% cells were induced apoptosis after 24h at concentration of 60, 120 and 180ug/ml respectively, relative to 3.9% of that of control group cell just only in the presence of UV.

Soluble Flk-1, a soluble vascular endothelial growth factor (VEGF) receptor, is a potent inhibitor of angiogenesis, which could restrain growth and metastasis of some experimental tumors. However, antiangiogenic agents alone cannot eradicate tumor completely, and should be combined with other therapy to enhance their effects. In this study, we evaluated the antitumor activity of the combination therapy in the immunocompetent BALB/c mice bearing H22 hepatoma and Meth A fibrosarcoma, respectively. Mice were treated with either msFlk-1 i.m. at 100 microg/mouse once every 3 days for four times from day 3 after the tumor cell injection, cisplatin cycled twice (2 mg/kg i.p. on days 4 and 11 after the tumor cell inoculation), or both agents together. Tumor growth and survival time were continually observed. Antiangiogenesis in vivo was determined by CD31 immunohistochemistry. Assessment of apoptotic cells and histological analysis was also conducted in tumor tissues. Our results showed that the combination therapy could evidently improve antitumor efficacy, including tumor growth suppression, mice survival prolongation, tumor cell apoptosis augmentation as well as neovascularization inhibition as compared with controls, without serious adverse effects. Our data suggest that the combination of DDP with msFlk-1 is more effective to suppress tumor growth in mice than either agent alone, and this combination regimen showed its potential for future clinical application.

The growth and persistence of solid tumors and their metastasis are angiogenesis-dependent. Vasostatin, the N-terminal domain of calreticulin inclusive of amino acids 1-180, is a potent angiogenesis inhibitor. To investigate whether intramuscular administration of vasostatin gene has the antitumor activity in mouse tumor models, we constructed a plasmid DNA encoding vasostatin and a control vector. Production and secretion of vasostatin protein by COS cells transfected with the plasmid DNA encoding vasostatin (pSecTag2B-vaso) were confirmed by Western blot analysis and ELISA. Conditioned medium from vasostatin-transfected COS cells apparently inhibited human umbilical vein endothelial cell (HUVEC) and mouse endothelial cell (SVEC4-10) proliferation, compared with conditioned medium from the COS cells transfected with control vector or non-transfected cells. Treatment with pSecTag2B-vaso twice weekly for 4 weeks resulted in the inhibition of tumor growth and the prolongation of the survival of tumor-bearing mice. The sustained high level of vasostatin protein in serum could be identified in ELISA. Angiogenesis was apparently inhibited in tumor by immunohistochemical analysis. Angiogenesis was also inhibited in the chicken embryo CAM assay and mouse corneal micropocket assay. The increased apoptotic cells were found within the tumor tissues from the mice treated with plasmid DNA encoding vasostatin. Taken together, the data in the present study indicate that the cancer gene therapy by the intramuscular delivery of plasmid DNA encoding vasostatin, is effective in the inhibition of the systemic angiogenesis and tumor growth in murine models. The present findings also provide further evidence of the anti-tumor effects of the vasostatin, and may be of importance for the further exploration of the application of this molecule in the treatment of cancer.

MicroRNAs (miRNAs), small and non-coding RNAs ∼ 22 nucleotides (nt) in length, are estimated to regulate about 30 % human gene expression at the post-transcriptional and the translational levels. MiRNAs are also involved in a series of important cellular processes, such as proliferation, differentiation, apoptosis and autophagy. Of note, apoptosis can invariably contribute to cell death, whereas autophagy can play either a pro-survival or a pro-death role in cancer. Recent evidence has shown that miRNAs can function as oncogenes or tumor suppressive genes in diverse types of human cancers. Also, miRNAs are well-characterized to be crucial in tumorigenesis as either oncogenes or tumor suppressors by targeting apoptosis and autophagy. However, the intricate mechanisms of miRNA-modulated apoptosis and autophagy still remain unclear. In this chapter, we focus on summarizing the dual function of miRNAs in regulation of apoptosis and autophagy; thereby revealing the regulatory mechanisms of miRNA-regulated apoptosis and autophagy, which may shed light on developing novel RNA therapeutic strategy in the near future.

Tumor cells often express elevated levels of heat-shock protein (HSP) 70. The present study was designed to investigate the role of HSP70 in the proliferation and survival of tumor cells in the human system. When Molt-4 and other tumor cells were treated in vitro with HSP70 antisense oligomer, they displayed propidium-iodide-stained condensed nuclei (intact or fragmented). A ladder-like pattern of DNA fragments was observed with HSP70 antisense-oligomer-treated tumor cells in agrose gel electrophoresis, which was consistent with internucleosomal DNA fragmentation. Flow cytometry analysis revealed the hypodiploid DNA peak of propidium-iodide-stained nuclei in the antisense-oligomer-treated cells. The apoptosis induced by HSP antisense oligomer was dose- and time-dependent. The antisense oligomer induced apoptosis mainly in tumor cells at G1 and S phase, resulting in an inhibition of cell proliferation. HSP70 antisense oligomer caused DNA-sequence-specific inhibition of HSP70 expression, which preceded apparent apoptosis. These results indicate that HSP70 antisense treatment inhibits the expression of HSP70, which in turn inhibits cell proliferation and induces apoptosis in tumor cells and suggest that HSP70 is required for tumor cells to proliferate and survive under normal condition.

To test the impact of silencing focal adhesion kinase (FAK) gene on human hepatocellular carcinoma cell line sk-hep-1 using RNA interference (RNAi) in vitro, and its therapeutic effect on human hepatocellular carcinoma xenograft in vivo.The plasmids generating short hairpin RNA that target FAK gene were transfected into sk-hep-1 cell line through Fugene HD. The impact was analyzed using flow cytometry, RT-PCR and western bolt. Human hepatocellular carcinoma xenograft in nude mice was established and treated with injection of the plasmids that packaged by cationic liposome into the tail veins the mice. The volumes and weights of the tumors were measured. The tumor tissues were analysed with FAK, CD31 and PCNA immunohistochemistry and TUNEL.The RNAi targeting FAK significantly down regulated the expression of FAK in mRNA and the level of protein (P0.001), and increased the cell apoptosis in vitro. The mice treated with pshFAK had slower tumor growth, resulting in smaller tumor size and lower tumor weight (P0.001) comparing to the controls. Decrease in FAK protein expression, tumor angiogenesis and proliferation, and increase in tumor apoptosis were found in the mice treated with pshFAK compared to the controls (P0.001).The shRNA targeting FAK suppresses the FAK expression of human hepatocellular carcinoma cells sk-hep-1, inhibits tumor growth, and promotes cell apoptosis in vitro and in vivo. FAK could become a candidate target for liver cancer treatment.

Deltonin, a steroidal saponin, isolated from Dioscorea zingiberensis Wright (DZW), has shown high-cytotoxic activity in cancer cells. However, its mechanisms and in vivo anti-cancer effects remain unknown. In the present study, we evaluated the effects and explored the anti-tumor mechanisms of deltonin on a panel of colon cancer cell lines and in a mouse model of murine colon cancer C26. Deltonin had more cytotoxic effect on C26 cells than 5-fluorouracil had, promoting dramatic G2-M phase arrest and apoptosis in C26 cells in a concentration-dependent manner; oral administration of deltonin significantly inhibited the tumor growth and prolonged survival of the tumor bearing mice. The deltonin treatment caused a noticeable apoptosis in tumor tissue, which associated with increased levels of Bax, activated caspase-3, caspase-9, and cleaved poly (ADPribose) polymerase, decreased pro-caspase-8, pro-caspase-9, Bcl-2 expression levels and extracellular signal-regulated kinase-1/2 activity; and dose-dependently inhibit angiogenesis. In conclusion, the findings in this study demonstrated that deltonin is an effective natural agent for cancer therapy, which may be mediated, in part, by induction of apoptosis, as well as involve mitogen-activated protein kinase pathways, and inhibition of angiogenesis.

Background and Objective: iASPP, an inhibitory member of the apoptosis-stimulating proteins of p53 (ASPP) family, has been found to be up-regulated in various human tumor types. This study was to construct an efficient doxycycline-regulated, lentiviral vector-mediated knockdown system for iASPP that will allow for inducible down-regulation of iASPP gene expression and preliminary functional analysis. Methods: A pair of complementary oligos with hairpin structures targeting the iASPP gene and a negative control were synthesized, then ligated with pLVTHM vector and sequenced. The fragment containing the shRNA cassette was cloned to pLVCT-tTR-KRAB plasmid. The recombinant vectors were co-transfected with viral packaging mix into 293T cells, and viral supernatant was harvested to determine the titer. After treatment with or without doxycycline, HepG2 cells infected with virus were harvested and the expression of iASPP was detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis. Its effects on tumor growth were characterized using MTS assay, soft agar colony formation, and flow cytometry analysis. Results: The lentiviral vector expressing shRNA that targets to the oncogene iASPP was constructed successfully. HepG2 infected with the lentivirus expressing 5hRNA against iASPP inhibited the expression of iASPP in the presence of doxycycline, which resulted in the repression of tumor cell proliferation and anchorage-independent growth potential. Conclusions: The lentiviral vector-mediated tet-on system demonstrates efficient and inducible knockdown of iASPP in hepatocellular carcinoma cells. iASPP gene may be involved in tumorigenesis and progression of human tumors.

Down-regulated in renal cell carcinoma gene (DRR1) is one of the candidate tumor suppressor genes (TSGs) on human 3p21.1. This study was performed to validate the expression status of DRR1 gene in cancer cells and the expression pattern of the protein in clinical specimens of human lung cancer and to examine its potential as a molecular target for treatment of lung cancer in vivo. DRR1 expression was analyzed in 7 human lung cancer cell lines. DRR1 protein expression was also examined in clinical non-small cell lung cancer (NSCLC) specimens. Furthermore, effects of DRR1 re-expression on A549 cells in vitro and A549 xenograft tumors in nude mice were evaluated. Loss of DRR1 mRNA expression was detected in 6 of the 7 human cancer cell lines, the exception was the renal cancer cell line OS-RC-2. DRR1 protein expression was absent in 15 of 20 (75%) human NSCLC specimens by immunostaining. Transfection of DRR1 gene into DRR1-negative-expressing A549 cells resulted in significant cell growth suppression and apoptosis. Plasmids containing DRR1 cDNA complexed with DOTAP:Chol liposomes were administered intravenously via tail vein to nude mice bearing A549 xenograft tumors resulting in tumor growth inhibition and elevation of apoptosis compared with the controls. DRR1 is a potent growth suppressor of NSCLC, acting through apoptosis pathway in vivo and it may be a potential therapeutic gene for human lung cancer.

To construct a eukaryotic co-expression plasmid containing FUS1 and IL-12, and to investigate the influence of the recombinant plasmid on the cultured human lung cancer cell line A549.RT-PCR was applied to amplify FUS1 from MRC-5 cell, the cDNA fragment of IL-12 was derived from pORF-hIL-12 by PCR, FUS1 cDNA fragment and IL-12 cDNA fragment were inserted into the multiple cloning sites of pVITRO2 to generate the eukaryotic co-expression plasmid pVITRO2-FUS1-IL-12. The recombinant plasmid was confirmed by restriction endonuclease treatment and sequenceing and transfected into A549 cells. The expression of genes in pVITRO2-FUS1-IL-12 were identified by RT-PCR, ELISA and Western blot. The function of pVITRO2-FUS1-IL-12 inducing the apoptosis of lung cancer was identified by Hoechst33258 and flow cytometry.The restriction endonuclease digestion and sequencing suggested that co-expression vector pVITRO2-FUS1-IL-12 was constructed successfully, the expression of FUS1 and IL-12 could be detected in A549 cells. The expressions of the two genes in pVITRO2 were not affected. The A549 cells transfected with pVITRO2-FUS1-IL-12 exhibited significant cell apoptosis.The recombinant eukaryotic expression vector pVITRO2-FUS1-IL-12 was constructed and expressed in eukaryotic cell successfully, which would contribute to a novel and effective treatment strategies for combined gene therapy for lung cancer.

To evaluate the anticancer effect of liposome plus Ad-Endostatin complex on human ovarian serous cystocarcinoma.The recombinant endostatin was expressed in the SKOV3 cells transfected with constructed adenovirus. The nude mice models with human ovarian serous cystocarcinoma were divided into six groups randomly: (1) Ad-hEndo-H plus liposome group (abbreviation: lipo+Ad-hEndo-H), i.v. administration of 1 x 10(9) pfu (plaque-forming units) recombinant adenovirus plus 200 microg liposome (n=5); (2) Ad-hEndo-L plus liposome group (abbreviation: lipo+Ad-hEndo-L), i.v. administration of 1 x 10(8) pfu recombinant adenovirus plus 20 microg liposome (n=5); (3) Ad-hEndo group, i.v. administration of 1 x 10(9) pfu recombinant adenovirus (n=4) (4) Ad-null plus liposome group, i.v. administration of 1 x 10(9) pfu adenovirus plus 200 microg liposome (n=4); (5) liposome group, i.v. administration of liposome 200 microg (n=4) (6) NS group, i.v. administration of equal volume of normal saline as above (n=4). The tumor size was monitored every 7 days. All of the nude mice were sacrificed 49 days after the tumor establishment. The tumors were removed and weighted. The micro-vessel density (MVD) was counted and the apoptotic cells were measured in the tumors tissue by TUNEL. The effect of the antibody of adenovirus was investigated with the SKOV3 cells transfected with liposome-complexed adenovirus.The tumors of the mice in the lipo+Ad-hEndo-H and lipo+Ad-hEndo-L groups weighted 54.74% and 70.65% lighter than the NS controls, respectively (P0.05). The tumors in the lipo+Ad-hEndo-L and lipo+Ad-hEndo-H groups had fewer MVD and more apoptotic cells than those in the other groups (P0.05). The antibody of adenovirus had less impact on the adenovirus capability of transfect when it was combined with liposome than without liposome.The liposome plus Ad-Endostatin complex inhibits the growth of human ovarian serous cystocarcinoma effectively. Lower dose of repeated injection of adenovirus with liposome is preferable.

To test the inhibitive effect of the matrix protein of vesicular stomatitis virus (VSV) on the proliferation of colon carcinoma cells of mice in vitro.The plasmid pcDNA3. 1-M encoding M protein of vesicular stomatitis viruses was transfected with lipofectamine 2000 into the colon carcinoma (CT26) cells of mice. The expression of VSV-M protein in the CT26 cells was detected by Western blot. The effect of vesicular stomatitis virus (VSV) matrix protein on the proliferation and apoptosis of colon carcinoma cells was measured by MTT, PI stainning and flow cytometry.The plasmid expressed M protein in the CT26 cells. Cytopathic effect was shown in the CT26 cells with transfected pcDNA3.1-M. The proliferation of the CT26 cells was suppressed significantly in vitro (76.4%, P0.05). The M protein induced apoptosis of the CT26 cells (80.2%), which was higher than'that of the controls (P0.05).The eukaryotic expression plasmid pcDNA3.1-M encoding M protein of vesicular stomatitis viruses induces apoptosis of CT26 cells, which has implications on the treatment of human colon cancer.

Epidemiological studies have demonstrated that high-risk human papillomavirus (HPV) is involved in causing cervical carcinoma. The HPV oncoproteins E6 and E7 immortalize human keratinocytes is mostly resulted from inactivation of tumor suppressor proteins p53 and pRB, which also play an important role in regulating the expression of pro- and antiangiogenic factors. The present study was conducted to determine whether IFN--inducible protein 10 (IP-10)/CXC chemokine ligand 10(CXCL10), one of the potent antiangiogenic chemokines, can inhibit the growth of cervical cancer. Plasmid DNA encoding CXCL10 was encapsulated with cationic liposomes, mice were treated with DNA-liposome mixture 6 times with the 5-day interval. Our results demonstrated that CXCL10 could reduce the level of HPV oncoproteins E6 and E7 in cervical cancer cells. In vivo study showed that CXCL10 could inhibit the growth of tumor in the immunodeficiency mice. Immunohistology analysis revealed that CXCL10 downregulated the microvessel density and the expression of PCNA in tumor tissues. TUNEL staining demonstrated CXCL10 significantly increase the apoptotic rate. Our data suggest that CXCL10 can inhibit the growth of cervical carcinoma through modulating the formation of microvessel and the expression of HPV oncoproteins E6 and E7. The present findings also provide further evidence of the anti-tumor effects of CXCL10, and may be of importance for the further exploration of the potential application of this molecule in the treatment of cervical carcinoma.

Background Bone marrow-derived stromal cells (BMSCs) are important for development, tissue cell replenishment, and wound healing in physiological and pathological conditions. BMSCs were found to preferably reach sites undergoing the process of cell proliferation, such as wound and tumor, suggesting that BMSCs may be used as a vehicle for gene therapy of tumor. Methods Mouse BMSCs were loaded with recombinant adenoviruses which express soluble Vascular Endothelial Growth Factor Receptor-1 (sFlt-1). The anti-angiogenesis of sFlt-1 in BMSCs was determined using endothelial cells proliferation inhibition assay and alginate encapsulation assay. The anti-tumor effects of BMSCs expressing sFlt-1 through tail-vein infusion were evaluated in two mouse tumor metastases models. Results BMSCs genetically modified with Adv-GFP-sFlt-1 could effectively express and secret sFlt-1. BMSCs loaded with sFlt-1 gene could preferentially home to tumor loci and decrease lung metastases and prolong lifespan in mouse tumor model through inducing anti-angiogenesis and apoptosis in tumors. Conclusion We demonstrated that BMSCs might be employed as a promising vehicle for tumor gene therapy which can effectively not only improve the concentration of anticancer therapeutics in tumors, but also modify the tumor microenvironment.

Chloroquine is an antimalarial drug that has been used in the treatment and prophylaxis of malaria since the 1950s. The present study was undertaken to examine the effects of chloroquine on Bcap-37 human breast cancer cells' growth, cell cycle modulation, apoptosis induction, and associated molecular alterations in vitro. The chloroquine treatment decreased the viability of Bcap-37 cells in a concentration- and time-dependent manner, which correlated with G(2)/M phase cell cycle arrest. The chloroquine-mediated cell cycle arrest was associated with a decrease in protein levels/activity of polo-like kinase 1 (Plk1), phosphorylated cell division cycle 25C (Cdc25C), phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2), phosphorylated Akt. The chloroquine-treated Bcap-37 cells exhibited a marked decrease in the level of mitochondrial transmembrane potential (DeltaPsim), which was accompanied by the activation of caspase-3 and cleaved poly(ADP-ribose) polymerase (PARP). Exposure of Bcap-37 cells to chloroquine also resulted in the induction of spindle abnormalities. In conclusion, the findings in this study suggested that chloroquine might have potential anticancer efficacy, which could be attributed, in part, to its proliferation inhibition and apoptosis induction of cancer cells through modulation of apoptosis and cell cycle-related proteins expressions, down-regulation of mitochondrial transmembrane potential (DeltaPsim), and induction of spindle abnormalities.

The purpose of this study was to investigate the effects of chloroquine diphosphate on the proliferation and apoptosis of human leukemic K562 cells, and to elucidate its possible mechanism of activity. The inhibitory effect of chloroquine diphosphate with different concentrations on K562 cell proliferation was detected by MTT method. Apoptosis was measured by flow cytometry (FCM); morphological analysis of apoptosis was performed after staining with propidium iodide (PI) under fluorescence microscope; cell apoptosis was assessed by the DNA ladder shown agarose gel electrophoresis. After treatment with chloroquine diphosphate, K562 cells were stained by Rhodamine 123 to detect changes in mitochondrial transmembrane potential (DeltaPsim) by FCM. The results showed that the cell viability decreased in dose-dependent manner, following chloroquine diphosphate treatment at different concentrations (1.5625, 3.125, 6.25, 12.5, 25, 50 and 100 micromol/L) for 24, 48 and 72 hours. By FCM analysis, the significant increases of sub-G(1) were observed. DNA ladder was detected and apoptotic nuclei were observed. DeltaPsim decreased in K562 cells after chloroquine diphosphate treatment. It is concluded that the chloroquine diphosphate can inhibit the proliferation of K562 cells and induce cell apoptosis, which may relate to down-regulation of mitochondrial transmembrane potential (DeltaPsim).

FAM92A1 (named FAM92A1-271) belongs to the family of proteins with conserved DUF1208 domains. Its function remains elusive. We identified two novel transcript variants (FAM92A1-251, FAM92A1-289) of FAM92A1. The presence of these transcripts in cancerous and normal cells, as well as their influence on cell proliferation and apoptosis, were investigated. The subcellular location of FAM92A1 was determined by fluorescence microscopy. We found that FAM92A1-271 and FAM92A1- 289 were highly expressed in both normal and cancerous cells, but FAM92A1-251 was only expressed at a moderate level in both types of cell. Overexpression of FAM92A1-271, FAM92A1-251 and FAM92A1-289 inhibited cell proliferation, caused S-phase arrest and induced apoptosis. Subcellular localization showed that FAM92A1 localizes to the nucleus. Our results show that FAM92A1 has different splicing variants, and that it may take part in regulating cell proliferation and apoptosis.

Quercetin is a potential chemotherapeutic drug with many biological activities. However, the insolubility of quercetin seriously limits its clinical use. This study was to investigate the biodistribution of quercetin encapsulated by pegylated nanoliposomes and its therapeutic efficacy on the formation of carcinomatous ascites of hepatocellular carcinoma in mice.Nanoliposomal quercetin was prepared with conventional rotary evaporation method. BALB/c mice inoculated with hepatocellular carcinoma cells (H22) at the anterior right subaxilla for twelve days were given intravascular injection with nanoliposomal quercetin at 1.5 mg/body (based on quercetin) at different time points. Then the levels of quercetin in the plasma, tumor tissues and normal organs were tested by high pressure liquid chromatography (HPLC). Various dosages of nanoliposomal quercetin were peritoneally given to tumor-bearing mice to determine the optimal dose. The tumor-bearing mice were treated intraperitoneally with 100 mg/kg nanoliposomal quercetin once a day for 14 days. The formation of malignant ascites, increase of body weight, survival time and peritoneal capillary permeability were assessed. Apoptotic cells in ascites were detected by flow cytometry.Nanoliposomal quercetin was a spherical particle with 25% drug content (W/W) and 130+/-20 nm in diameter. Nanoliposomal quercetin effectively aggregated in tumor tissues and its half-life period was 4 h. Nanoliposome quercetin inhibited the formation of malignant ascites of hepatocellular carcinoma model in a dose-dependent manner. Moreover, 100 mg/kg nanoliposomal quercetin significantly enhanced the apoptosis of cancer cells in ascites, inhibited the increase of body weight, reduced peritoneal capillary permeability and prolonged the survival time of tumor-bearing mice compared with PBS control.Nanoliposomal quercetin can effectively accumulate in tumor tissues and inhibit the formation of malignant ascites, thus it might be used as a potential antitumor drug which deserves future study.

MIG (monokine induced by interferon-gamma) is a CXC chemokine ligand (CXCL9) that can potently inhibit angiogenesis, and displays thymus-dependent antitumor effects. The effectiveness of a treatment combining gene therapy with plasmid-borne MIG (pORF-MIG) and low-dose cisplatin chemotherapy was determined using colon carcinoma (CT26) and Lewis lung carcinoma (LL/2c) murine models. The program was carried out via intramuscular delivery of pORF-MIG at 100 mug/mouse twice a week for 4 weeks, and/or intraperitoneal delivery of cisplatin at 0.6 mg/kg/mouse every 3 days for 48 days. Tumor volume and survival time were evaluated after treatment. CD31 immunohistochemical staining in tumor tissues and alginate capsule models in vivo was used to evaluate angiogenesis. Induction of apoptosis and cytotoxic T-lymphocyte (CTL) activity were also assessed. The combination of pORF-MIG and low-dose cisplatin produced significant antitumor activity, with complete tumor regression in 4/10 of CT26 colon carcinomas and 3/10 of LL/2c lung carcinomas, low vascularity, in alginate capsules, apparently degraded tumor microvessel density, and increased induction of apoptotic and CTL activities compared with either treatment alone. This study suggests that the combination of pORF-MIG plus cisplatin augments the inhibition of angiogenesis and the induction of apoptosis or CTL activity, all of which enhance antitumor activity. These findings may prove useful in further explorations of the application of combinatorial approaches to the treatment of solid tumors.

To evaluate the effect of full-length cyclin B1 antisense cDNA (AS-CLB1) on chemosensitivity of Lewis lung carcinoma cells (LL/2) to gemicitabine (GEM) in vitro and in vivo and hence provide a therapeutic regiment for treating non-small cell lung (NSCL) cancer using AS-CLB1 combined with GEM.Cell cycle phase distribution and apoptosis of LL/2 parent cells, LL/2/vect and LL/2/AS-CLB1 transfectants (LP, LV and LA cells) were determined by flow cytometry. In addition, the three kinds of cells were treated with GEM (20 nmol/L-20 micromol/L) in vitro for 1 h and 24 h respectively, and then cytotoxicity of GEM was measured by MTT assay. After inoculation with the three kinds of cells respectively, the C57BL/6 mice were treated with GEM [25 - 125 mg/(kg x day)] once every three days for four times when tumors developed; tumorigenicity and survival were observed and cell apoptosis in tumor tissues was determined by flow cytometry.LA cells displayed apparent apoptosis and G1 arrest compared with LP and LV cells (controls). Additionally, cytotoxicity of GEM to LA cells was more obvious than that to controls. Moreover, tumorigenicity was inhibited, cell apoptosis in tumor tissues was induced, and survival was evidently increased in LA cells group.AS-CLB1 slightly increased the sensitivity of LL/2 cells to GEM in vitro and in vivo. The function of AS-CLB1 may be associated with its ability to enhance the anti-tumor activity of GEM by inducing cell apoptosis and G1 arrest.

Interferon-gamma-inducible protein 10 (IP-10), a member of alpha-chemoattractant family, is an inhibitor of tumor angiogenesis. Chemotherapy combined with antiangiogenic therapy has synergistic effects. This study was designed to explore antitumor effect of IP-10 combined with gemcitabine in tumor-bearing mice.pBLAST-IP-10 was transferred into E.coli to prepare and purify plasmid IP-10. Hepatocarcinoma H22 model was established in BALB/c miceu Lewis lung cancer LL/2 model was established in C57BL/6 mice. The mice were divided into 4 groups, and treated with IP-10, gemcitabine, IP-10 plus gemcitabine, and normal saline (control), respectively. Protein level of IP-10 in serum was detected by ELISA. Survival rate of mice, tumor volume, and side effects of treatment were observed. Microvessel density (MVD) of tumor tissue was detected by immunohistochemistry. Apoptosis of tumor cells was detected by TUNEL method.After treatment of IP-10, IP-10 protein reached the peak level of (16.8+/-3.6) ng/ml at the 14th day, and remained a high level of (14.0+/-2.1) ng/ml at the 35th day. Compared with the controls, tumor volume of mice in the combination therapy group had significant regression, or disappeared. Nine weeks after inoculation of tumor cells, survival rate of mice was significantly higher in combination group than in IP-10, gemcitabine, and control groups (90% vs. 55%, 0%, and 0%, P0.005). No obvious side effects were observed. MVD of tumor tissues was significantly lower in combination group than in IP-10, gemcitabine, and control groups (15.8+/-2.4 vs. 45.6+/-2.0, 50.2+/-3.5, and 51.3+/-3.0, P0.01). At the 35th day after inoculation of tumor cells, apoptosis index was significantly higher in combination group than in IP-10, gemcitabine, and control groups (85.5+/-10.2 vs. 21.4+/-5.5, 8.4+/-2.0, 4.2+/-0.7, P0.01).Therapy of IP-10 combined with gemcitabine has significantly synergistic antitumor effect compared with IP-10 or gemcitabine alone.

Chemokines are small molecule proteins that are the main mediators of cell migration. Studies indicated that chemokines could induce antitumor immune response strongly and steadily. At present, there was not antitumor therapy about chemokine BLC combined with chemotherapy. This study was designed to explore the anti-tumor effects and mechanisms of BLC-modified tumor cell vaccine combined with cisplatin.Firstly, we transfected pcDNA-BLC into murine tumor cell lines and established stable transfected tumor cell lines (Colon26 and LL/2) to express BLC. Null plasmid pcDNA3.1 (+) was also transfected into Colon 26 or LL/2 cells stably for comparison. We established mouse model with pcDNA-BLC stable transfected tumor cells (Tc), control mouse model with pcDNA3.1(+) stable transfected tumor cells (Pc), and mouse model with parental tumor cells (Nc). Then the 60 mice were inoculated subcutaneously (s.c.) in the right flanks with a total of 5x10(5) viable tumor cells (Tc to 20 mice, Pc to 20 mice, and Nc to 20 mice, respectively; 6-8 week BALB/c female mouse in colon 26 mouse model or 6-8 week C57BL/6 female mouse in LL/2 mouse model). The groups (Tc, Pc, and Nc) were randomly subdivided into Group A and Group B with 10 mice for each, resulting in Tc-A/Tc-B, Pc-A/Pc-B, and Nc-A/Nc-B, respectively. Group A (including Tc-A, Pc-A, and Nc-A) as chemotherapy group was injected (i.p.) with 0.1 ml cisplatin at the concentration of 2 mg/kg once a week for two weeks; Group B (including Tc-B, Pc-B, and Nc-B) as control with 0.1 normal saline. We further observed anti-tumor activity including the tumor growth, mice survival rate, side effects as well as tumor morphological analysis. Apoptotic cells were also determined in tumor tissues.The combination therapy group Tc-A showed significant anticancer activities. The tumor growth was inhibited efficiently with 3/10 mice showing complete regression in LL/2 mouse model and 1/10 mice showing complete regression in Colon 26 mouse model. The combination therapy group Tc-A showed that the survival rate of mice was 100% in the two mouse models, compared with 68% in group Tc-B in C57BL/6 mouse model and 65% in group Tc-B in Colon 26 mouse model and those in other groups. In addition, the combination therapy group Tc-A induced tumor cell apoptosis significantly.The therapy of BLC-modified tumor cell vaccine combined with cisplatin had significant synergistic effect against tumor. It might develop a new approach for specific immunotherapy of tumors.

Tanshinone IIA is a derivative of phenanthrene-quinone isolated from Salvia miltiorrhiza BUNGE, which is a traditional herbal medicine that is used to treat cardiovascular diseases. Recent studies showed that Tanshinone IIA possesses cytotoxic activity against many kinds of human carcinoma cell lines, induces differentiation and apoptosis and inhibits invasion and metastasis of cancer cells. Its mechanisms are thought as inhibiting DNA synthesis and proliferation of cancer cells, regulating the expression of genes related to proliferation, differentiation, and apoptosis, inhibiting the telomerase activity of cancer cells, and changing the expression of cellular surface antigen.

Purpose: Cantharidin, a natural toxin, is the active substance of mylabris and has antitumor effects in man. Norcantharidin, the demethylated analogue of cantharidin, has been used in the treatment of patients with primary hepatoma and those with leukopenia in China. The present study was designed to investigate whether norcantharidin exerts cytotoxic activity against colorectal cancer cells by inducing apoptosis and to examine the possible mechanism in the phenomenon. Methods: Inhibition of proliferation of norcantharidin on Colo205, HT-29, and SW480 colorectal cancer cells was determined by the trypan blue dye exclusion test. Apoptosis of norcantharidin-treated cells was determined by morphological analysis, agarose gel DNA electrophoresis, and quantitated by flow cytometry after staining with propidium iodide. Cell cycle and the cell surface expression of the CD95/CD95 ligand were evaluated by flow cytometry. Caspase 8-like protease and protein phosphatase 1 and 2A activities were also analyzed. Results: Treatment with norcantharidin of colorectal cancer cells not only inhibited cell proliferation, but also induced apoptosis. Norcantharidin induced apoptosis mainly in two phases: rapid apoptosis in S-phase cells and delayed apoptosis in G2/M arrested cells. Treatment with norcantharidin resulted in an upregulation of the CD95 receptor and CD95 ligand on the cell surface. Furthermore, stimulation with anti-CD95 monoclonal antibody (mAb) resulted in further induction of apoptosis after treatment with norcantharidin. In addition, the apoptosis-inducing effect of norcantharidin was almost completely inhibited by anti-CD95 ligand mAb. Norcantharidin-treated cells showed the activation of caspase 8. Both zVAD-FMK (a broad range caspase inhibitor) and IETD-FMK (a caspase-8 inhibitor) showed apparent inhibition of the apoptosis-inducing effect. Norcantharidin did not show an inhibitory effect on protein phosphatase. Conclusions: These results suggest that norcantharidin triggers apoptosis in colorectal cancer cell lines via the activation of the CD95 receptor/ligand system, and that this agent may be useful for developing new therapeutic regimens for the treatment of colorectal carcinoma.

Honokiol is an active compound purified from magnolia that has been shown to induce cell differentiation, apoptosis, and anti-angiogenesis effects, as well as an enhancement in tumor growth delay in combination with chemotherapeutic agents in several mouse xenograft models. Our goal was to investigate the radiosensitization effect of honokiol on lung carcinoma. The radiosensitization effect of liposomal honokiol in Lewis lung carcinoma cells (LL/2) was analyzed using an in vitro clonogenic survival assay. For an in vivo study, Lewis lung carcinoma-bearing C57BL/6 mice were treated with either liposomal honokiol at 25 mg/kg or 5 Gy of single tumor radiation, or a combination of both over 12 days of treatment. The tumor growth delay and the survival time were evaluated. In addition, histological analysis of tumor sections was performed to examine changes by detecting the microvessel density and apoptosis in tumor tissues. In the clonogenic survival assay, LL/2 cells treated with IC(50) Lipo-HNK for 24 h showed a radiation enhancement ratio of 1.9. After 12 days of combination treatment, the tumor volume decreased 78% and produced an anti-tumor activity 1.3-fold greater than a predicted additive effect of honokiol and radiation alone. This combination treatment also caused an 8.7 day delay in tumor growth. The cell cycle distribution and histological analysis demonstrated that liposomal honokiol has an anti-tumor effect via inducing apoptosis and inhibiting angiogenesis. Liposomal honokiol can enhance tumor cell radiosensitivity in vitro and in vivo, indicating that radiotherapy combined with liposomal honokiol can lead to greater anti-tumor efficacy.