Targeted deletion of miR-139-5p activates MAPK, NF-κB and STAT3 signaling and promotes intestinal inflammation and colorectal cancer.

miR-139-5p, which has been reported to be underexpressed in several types of cancer, is associated with tumorigenesis by participating in various biological processes via the modulation of different target genes. In the present study, we analyzed mice deficient in miR-139-5p, aiming to investigate its role in intestinal inflammation and colitis-associated colorectal cancer. We show that miR-139-5p knockout (KO) mice are highly susceptible to colitis and colon cancer, accompanied by elevated proliferation and decreased apoptosis, as well as an increased production of inflammatory cytokines, chemokines and tumorigenic factors. Furthermore, enhanced colon inflammation and colorectal tumor development in miR-139-5p KO mice are a result of the regulatory effects of miR-139-5p on its target genes for Rap1b and nuclear factor-kappa B, thus affecting the activity of the mitogen-activated protein kinase, nuclear factor-kappa B and signal transducer and activator of transcription 3 signaling pathways. These results reveal a critical part for miR-139-5p in maintaining intestinal homeostasis and protecting against colitis and colorectal cancer in vivo, providing new insights into the function of miR-139-5p with respect to linking inflammation to carcinogenesis.

Photodynamic-therapy Activates Immune Response by disrupting Immunity Homeostasis of Tumor Cells, which Generates Vaccine for Cancer Therapy.

Photodynamic therapy (PDT), a regulatory approved cancer treatment, is reported to be capable of causing immunogenic apoptosis. The current data reveal PDT can cause the dysregulation of "eat me" and "don't eat me" signal by generating reactive oxygen species (ROS) -mediated endoplasmic reticulum (ER) stress. This dysregulation probably contribute to the increased uptake of PDT-killed Lewis lung carcinoma (LLC) cells by homologous dendritic cells (DCs), accompanied by phenotypic maturation (CD80(high), CD86(high), and CD40(high)) and functional stimulation (NO(high), IL-10(absent)) of dendritic cells as well as subsequent T-cell responses. Morevover, C57BL/6 mice vaccinated with dendritic cells (DCs) pulsed with PDT-treated LLCs (PDT-DCs) or PDT-treated LLCs alone (PDT-LLCs) exhibited potent immunity against LLC tumors. In the current study, the PDT-induced immune response was characterized as a process related with the dysregulation of "eat me" signal and "don't eat me" signal, revealing the possibility for developing PDT into an antitumor vaccination strategy for personalized cancer immunotherapy.

An investigation of antitumor efficiency of novel sustained and targeted 5-fluorouracil nanoparticles.

Traditional chemotherapeutic drugs remain the major treatment for advanced colorectal cancer. However, due to the lack of tumor specificity these drug also destroy healthy tissue and organs, which has been the main reason for treatment failure and mortality. Folate-based drug delivery systems for improving nanoparticle endocytosis have been used to address these problems. Here, folic acid (FA) conjugated mPEG-b-P(CABCL-co-ACL) diblock copolymers were synthesized and characterized by TEM and NMR. Drug loaded nanoparticles were prepared using dialysis method and was obtained with a mean diameter of 45.2 nm with sustained in vitro release profile. In vitro cytotoxicity assay indicated that the cytotoxicity of folate modified nanoparticles were significantly increased compared to free drug and non-folate nanoparticles. In addition, results of hemolytic and histopathologic study suggested that the non-loaded nanoparticle (NL/NP) was non-toxic and biocompatible at the testing concentration. Moreover, in vivo results showed that FA/5-FU/NP effectively inhibited growth of HCT-8 cell-based xenograft tumors in BALB/c mice and revealed stronger antitumor efficacy than other treated groups. Thus, both in vitro and in vivo results exhibited that the folate conjugated mPEG-b-P(CABCL-co-ACL) copolymers have great potential to be used as sustainable and specific colon cancer targeting delivery system for anticancer agents.

3B, a novel of photosensitizer, exhibited anti-tumor effects via mitochondrial apoptosis pathway in MCF-7 human breast carcinoma cells.

Photodynamic therapy (PDT) has been recognized as an innovated therapeutic modality for the treatment of various cancers. In this study, we evaluated the anticancer effect of a new photosensitizer 3B in breast cancer, which was considered one of the most common cancers in women worldwide. Here, we determined the effect of 3B not only on the cell growth, apoptosis, and Bcl-2 signal pathway in vitro but also on the anti-cancer effect in nude mice in vivo. Our results showed that 3B was primarily accumulated in mitochondria, increased the level of ROS, induced apoptotic cells death via Bcl-2 family, and its activity could be blocked by the caspase inhibitor (Z-VAD-FMK). In vivo study, 3B made a significant opening inhibition of tumor growth and showed drug toxicity hardly. TUNEL assay indicated that PDT group showed more positive cells (green) than other groups. These data supported that 3B might develop as potential therapeutic drug for the treatment of breast cancer.

Photodynamic therapy-mediated cancer vaccination enhances stem-like phenotype and immune escape, which can be blocked by thrombospondin-1 signaling through CD47 receptor protein.

Like most of the strategies for cancer immunotherapy, photodynamic therapy-mediated vaccination has shown poor clinical outcomes in application. The aim of this study is to offer a glimpse at the mechanisms that are responsible for the failure based on cancer immuno-editing theory and to search for a positive solution. In this study we found that tumor cells were able to adapt themselves to the immune pressure exerted by vaccination. The survived tumor cells exhibited enhanced tumorigenic and stem-like phenotypes as well as undermined immunogenicity. Viewed as a whole, immune-selected tumor cells showed more malignant characteristics and the ability of immune escape, which might contribute to the eventual relapse. Thrombospondin-1 signaling via CD47 helped prevent tumor cells from becoming stem-like and rendered them vulnerable to immune attack. These findings prove that the TSP-1/CD47/SIRP-α signal axis is important to the evolution of tumor cells in the microenvironment of immunotherapy and identify thrombospondin-1 as a key signal with therapeutic benefits in overcoming long term relapse, providing new evidence for the clinical promise of cancer vaccination.

Ganoderic acid Me induces the apoptosis of competent T cells and increases the proportion of Treg cells through enhancing the expression and activation of indoleamine 2,3-dioxygenase in mouse lewis lung cancer cells.

The indoleamine 2,3-dioxygenase-(IDO-) mediated microenvironment plays an important role in tumor immune escape. It is known that ganoderic acid Me can enhance IFN-γ expression and IDO is preferentially induced by IFN-γ. However, whether GA-Me can induce IDO expression has not been clarified yet. We established stable clones of IDO-overexpressing 2 LL cells (2LL-EGFP-IDO). After co-culturing with IDO expressing or control vector-transfected 2LL-EGFP cells, T cell apoptosis was determined and the proportion of the regulatory T cells (Tregs) and CD8+ T cell subset was measured. The total cellular protein samples of 2 LL-EGFP-IDO cells were isolated for detecting JAK-STAT1 signalling pathway. Co-culture supernatants were used to detect amino acids and cytokines. IDO transfected 2 LL cells yielded high level of IDO enzymatic activity, resulting in complete depletion of tryptophan from the culture medium. We found that apoptosis occurred in T cells after cocultured with IDO+2LL cells and the proportion of CD4+CD25+ cells and FoxP3+ cells increased while CD8+ cells decreased. The specific inhibitor of IDO, 1-D-MT and GA-Me efficiently enhanced T cell apoptosis, increased Tregs, and reduced CD8+ T cells in vitro. Increased expression of IDO, p-JAK1 and p-STAT1 were confirmed by Western blot analysis. The levels of IFN-γ, IL-10, LDH and kynurenine in co-culture supernatant correspondingly increased, while tryptophan reduced. These results suggest that GA-Me contributing to IDO helps to create a tolerogenic milieu in lung tumors by directly inducing T cell apoptosis, restraining CD8+ T cell activation, and enhancing Treg-mediated immunosuppression.

Astragaloside IV inhibits progression of lung cancer by mediating immune function of Tregs and CTLs by interfering with IDO.

PURPOSE: Tumor cells have developed multiple mechanisms to escape immune recognition mediated by T cells. Indoleamine 2,3-dioxygenase (IDO), a tryptophan-catabolizing enzyme inducing immune tolerance, is involved in tumor escape from host immune systems in mice. Astragaloside IV (AS-IV), an extract from a commonly used Chinese medicinal plant Astragalus membranaceus, has been shown to be capable of restoring the impaired T-cell functions in cancer patients. The purpose of this study was to investigate the mechanisms underlying the anticancer properties of AS-IV. METHODS: Here, we used IDO-overexpressed murine Lewis lung carcinoma cells to establish an orthotopic lung cancer model in C57BL/6 mice. Next, tumor growth was evaluated in several different treatment groups: control (saline), AS-IV, paclitaxel, and 1-methyl tryptophan (an inhibitor of IDO). We then analyzed the percentages of various immune cell subsets among the splenic lymphocytes of lung cancer mice by flow cytometry. The level of IDO was measured by real-time PCR and Western blot. RESULTS: We showed that the growth of tumor was suppressed by AS-IV treatment in vivo. AS-IV also could down-regulate regulatory T cells (Tregs) and up-regulate cytotoxic T lymphocytes (CTLs) in vivo and in vitro. Consistent with its ability to interfere with T-cell immunity, AS-IV blocked IDO induction both in vitro and in vivo. CONCLUSIONS: The results of these studies indicate that AS-IV has in vivo anticancer activity and can enhance the immune response by inhibiting the Tregs frequency and induce the activity of CTLs, which might be related to the inhibition of IDO expression.

Atractylenolide I-mediated Notch pathway inhibition attenuates gastric cancer stem cell traits.

Atractylenolide I (AT-I), one of the main naturally occurring compounds of Rhizoma Atractylodis Macrocephalae, has remarkable anti-cancer effects on various cancers. However, its effects on the treatment of gastric cancer remain unclear. Via multiple cellular and molecular approaches, we demonstrated that AT-I could potently inhibit cancer cell proliferation and induce apoptosis through inactivating Notch pathway. AT-I treatment led to the reduction of expressions of Notch1, Jagged1, and its downstream Hes1/ Hey1. Our results showed that AT-I inhibited the self-renewal capacity of gastric stem-like cells (GCSLCs) by suppression of their sphere formation capacity and cell viability. AT-I attenuated gastric cancer stem cell (GCSC) traits partly through inactivating Notch1, leading to reducing the expressions of its downstream target Hes1, Hey1 and CD44 in vitro. Collectively, our results suggest that AT-I might develop as a potential therapeutic drug for the treatment of gastric cancer.

Overcoming multidrug resistance in 2D and 3D culture models by controlled drug chitosan-graft poly(caprolactone)-based nanoparticles.

The principal limitations of chemotherapy are dose-limiting systemic toxicity and the development of multidrug-resistant phenotypes. The aim of this study was to investigate the efficiency of a new sustained drug delivery system based on chitosan and ε-caprolactone to overcome multidrug resistance in monolayer and drug resistance associated with the three-dimensional (3D) tumor microenvironment in our established 3D models. The 5-fluorouracil (5-FU)-loaded nanoparticles (NPs) were characterized by transmission electron microscope and dynamic light scattering, and its released property was determined at different pH values. 5-FU/NPs exhibited well-sustained release properties and markedly enhanced the cytotoxicity of 5-FU against HCT116/L-OHP or HCT8/VCR MDR cells in two-dimensional (2D) and its parental cells in 3D collagen gel culture with twofold to threefold decrease in the IC50 values, as demonstrated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, Hoechst/propidium iodide staining and flow cytometry analysis. Furthermore, the possible mechanism was explored by high-performance liquid chromatography and rhodamine 123 accumulation experiment. Overall, the results demonstrated that 5-FU/NPs increase intracellular concentration of 5-FU and enhance its anticancer efficiency by inducing apoptosis. It was suggested that this novel NPs are a promising carrier to decrease toxic of 5-FU and has the potential to reverse the forms of both intrinsic and acquired drug resistance in 2D and 3D cultures.

Hypericin-based Photodynamic Therapy Induces a Tumor-Specific Immune Response and an Effective DC-based cancer Immunotherapy.

In our study, we find that photodynamic therapy (PDT), which generates reactive oxygen species (ROS) -mediated endoplasmic reticulum (ER) stress to inflict trauma in the targeted lesion, can break the balance between membrane damage-associated molecular patterns (DAMPs) and integrin-associated protein (CD47). The imbalance undermines the ability of lewis lung carcinoma (LLC) cells to escape immune attack by increasing the uptake of hypericin-mediated PDT(hyp-PDT) killed lewis lung carcinoma (LLC) cells by homologous dendritic cells (DCs), accompanied by phenotypic maturation (CD80high, CD86high, and CD40high) and functional stimulation (NOhigh, IL-10absent) of dendritic cells as well as subsequent T-cell response. Besides, C57BL/6 mice vaccinated with dendritic cells (DCs) pulsed with PDT-treated LLCs (PDT-DCs) or PDT-treated LLCs alone (PDT-LLCs) show potent immunity against LLC tumor. These data identify hypericin-induced PDT as a strong inducer of immunogenic apoptosis, providing an antitumor vaccination strategy for personalized cancer Immunotherapy.

Development of rapid and highly sensitive HSPA1A promoter-driven luciferase reporter system for assessing oxidative stress associated with low-dose photodynamic therapy.

Photodynamic therapy (PDT) is a regulatory-approved modality for treating a variety of malignant tumors. It induces tumor tissue damage via photosensitizer-mediated oxidative cytotoxicity. The heat shock protein 70 (HSP70-1) is a stress protein encoded by the HSPA1A gene and is significantly induced by oxidative stress associated with PDT. The aim of this study was to identify the functional region of the HSPA1A promoter that responds to PDT-induced oxidative stress and uses the stress responsiveness of HSPA1A expression to establish a rapid and cost-effective photocytotoxic assessment bioassay to evaluate the photodynamic potential of photosensitizers. By constructing luciferase vectors with a variety of hspa1a promoter fractions and examining their relative luciferase activity, we demonstrated that the DNA sequence from -218 to +87 of the HSPA1A gene could be used as a functional promoter to detect the PDT-induced oxidative stress. The maximal relative luciferase activity level of HSPA1A (HSP70-1) induced by hypericin-PDT was nearly nine times that of the control. Our results suggest that the novel reporter gene assay using a functional region of the HSP70A1A promoter has significant advantages for the detection of photoactivity in terms of both speed and sensitivity, when compared with a cell viability test based on ATP quantification and ROS levels. Furthermore, phthalocyanine zinc and methylene blue both induced significantly elevated levels of relative luciferase activity in a dose-dependent manner.

miR-297 modulates multidrug resistance in human colorectal carcinoma by down-regulating MRP-2.

Colorectal carcinoma is a frequent cause of cancer-related death in men and women. miRNAs (microRNAs) are endogenous small non-coding RNAs that regulate gene expression negatively at the post-transcriptional level. In the present study we investigated the possible role of microRNAs in the development of MDR (multidrug resistance) in colorectal carcinoma cells. We analysed miRNA expression levels between MDR colorectal carcinoma cell line HCT116/L-OHP cells and their parent cell line HCT116 using a miRNA microarray. miR-297 showed lower expression in HCT116/L-OHP cells compared with its parental cells. MRP-2 (MDR-associated protein 2) is an important MDR protein in platinum-drug-resistance cells and is a predicted target of miR-297. Additionally miR-297 was down-regulated in a panel of human colorectal carcinoma tissues and negatively correlated with expression levels of MRP-2. Furthermore, we found that ectopic expression of miR-297 in MDR colorectal carcinoma cells reduced MRP-2 protein level and sensitized these cells to anti-cancer drugs in vitro and in vivo. Taken together, our findings suggest that miR-297 could play a role in the development of MDR in colorectal carcinoma cells, at least in part by modulation of MRP-2.