The role of angiotensin-(1-7) on acquired platinum resistance-induced angiogenesis in non-small cell lung cancer in vitro and in vivo.

Renin-angiotensin system (RAS) signaling has been implicated in the development of cancer. The new RAS ACE2/Ang-(1-7)/Mas axis antagonizes the classical ACE/Ang II/AT1R axis. Ang-(1-7) has pleiotropic roles in lung cancer including suppressing proliferation, angiogenesis, and metastasis. This research was designed to investigate the effect of Ang-(1-7) on tumor-associated angiogenesis in DDP-resistant lung cancer cell lines. We first established acquired DDP-resistant cell lines A549 (A549-DDP) and LLC (LLC-DDP). We next performed RT-qPCR, western blot, ELISA, tube formation, microvessel density detection, immunohistochemistry, and tumor formation assays. The results showed that the mRNA and protein levels of RAS components and vascular endothelial growth factor A (VEGFa) were lessened in the A549/LLC-DDP+Ang-(1-7) group compared with the A549/LLC-DDP group. This effect could be blocked by the MAS receptor antagonist A779. The data revealed that Ang-(1-7) could perform its antiangiogenic function by PI3K/AKT and MAPK pathways. Furthermore, the impact of Ang-(1-7) on tumor-associated angiogenesis has been confirmed in lung cancer xenograft model with acquired DDP resistance. These results provide a theoretical basis for designing therapeutic strategies for targeting Ang-(1-7) in the treatment of NSCLC.

Bufalin reverses multidrug resistance by regulating stemness through the CD133/nuclear factor-κB/MDR1 pathway in colorectal cancer.

Recent studies have shown that MDR could be induced by the high stemness of cancer cells. In a previous study, we found bufalin could reverse MDR and inhibit cancer cell stemness in colorectal cancer, but the relationship between them was unclear. Here we identified overexpressing CD133 increases levels of Akt/nuclear factor-κB signaling mediators and MDR1, while increasing cell chemoresistance. Furthermore, bufalin reverses colorectal cancer MDR by regulating cancer cell stemness through the CD133/nuclear factor-κB/MDR1 pathway in vitro and in vivo. Taken together, our results suggest that bufalin could be developed as a novel 2-pronged drug that targets CD133 and MDR1 to eradicate MDR cells and could ultimately be combined with conventional chemotherapeutic agents to improve treatment outcomes for patients with colorectal cancer.

Targeting CD133 reverses drug-resistance via the AKT/NF-κB/MDR1 pathway in colorectal cancer.

BACKGROUND: Recent studies have shown that multidrug resistance may be induced by the high stemness of cancer cells. Following prolonged chemotherapy, MDR protein 1 (MDR1) and CD133 increase in CRC, but the relationship between them is unclear. METHODS: The relationship between MDR and CSC properties in CRC was determined via CCK-8 assay, apoptosis assay, DOX uptake and retention, immunohistochemistry, immunofluorescence and flow cytometry. The correlations between their expression levels were evaluated using Spearman's rank statistical test and the Mann-Whitney test. Furthermore, the effect of CD133 on the repression of the AKT/NF-κB/MDR1 signalling pathway was investigated in vitro and in vivo. RESULTS: We found that CD133 increased with the emergence of drug-resistance phenotypes, and the high expression of MDR1/P-gp was consistently accompanied by positive expression of CD133 as demonstrated by the analysis of patient samples. Up- or downregulation of CD133 could regulate MDR via AKT/NF-κB/MDR1 signalling in CRC. A rescue experiment showed that the AKT/NF-κB signalling pathway is the main mechanism by which CD133 regulates MDR1/P-gp expression in CRC. CONCLUSIONS: Taken together, our results suggest that targeting CD133 reverses drug resistance via the AKT/NF-κB/MDR1 pathway and that this pathway might serve as a potential therapeutic target to reverse MDR in CRC.

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.

Discovery of novel 1,5-benzodiazepine-2,4-dione derivatives as potential anticancer agents.

A series of novel 1,5-benzodiazepine-2,4-dione derivatives with C-6 amide substituents were designed and synthesized using three-component reactions. The preliminary assays showed that most of them displayed moderate to good antitumor activities against human lung carcinoma (A549), human breast epithelial carcinoma (MCF-7), human colon carcinoma (HCT116), human cervical carcinoma (Hela) and Lewis lung carcinoma (2LL). Exhilaratingly, the activity level of 6m rivaled that of 5-Fluorouracil (5-Fu) against MCF-7 cell lines, which might be used as novel lead scaffold for potential anticancer development.

A collagen-based multicellular tumor spheroid model for evaluation of the efficiency of nanoparticle drug delivery.

Targeted drug delivery systems, especially those that use nanoparticles, have been the focus of research into cancer therapy during the last decade, to improve the bioavailability and delivery of anticancer drugs to specific tumor sites, thereby reducing the toxicity and side effects to normal tissues. However, the positive antitumor effects of these nanocarriers observed in conventional monolayer cultures frequently fail in vivo, due to the lack of physical and biological barriers resembling those seen in the actual body. Therefore, the collagen-based 3-D multicellular culture system, to screen new nanocarriers for drug delivery and to obtain more adequate and better prediction of therapeutic outcomes in preclinical experiments, was developed. This 3-D culture model was successfully established using optimized density of cells. Our result showed that 3-D cell colonies were successfully developed from 95-D, U87 and HCT116 cell lines respectively, after a seven-day culture in the collagen matrix. The coumarin-conjugated nanoparticles were able to penetrate the matrix gel to reach the tumor cells. The model is supposedly more accurate in reflecting/predicting the dynamics and therapeutic outcomes of candidates for drug transport in vivo, and/or investigation of tumor biology, thus speeding up the pace of discovery of novel drug delivery systems for cancer therapy.

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.

Preparation of polysaccharide derivates chitosan-graft-poly(ɛ-caprolactone) amphiphilic copolymer micelles for 5-fluorouracil drug delivery.

Biodegradable graft copolymer, chitosan-graft-poly(ɛ-caprolactone) (CS-g-PCL) was synthesized via ring opening polymerization and characterized by (1)H NMR and FTIR spectroscopy. Then graft copolymers were self-assembled into micelles as drug delivery system. To evaluate drug-polymer compatibility, the Flory-Huggins interaction parameter between 5-fluorouraci (5-Fu) and hydrophobic segment was calculated. The result was in agreement with experimental data from drug loading content and drug loading efficiency. Meanwhile, DLS and TEM were utilized to evaluate the trend of particle size and morphology in aqueous solution with different repeating units of ɛ-CL. The in vitro drug release data was fitted with three kinetic models, usually applied in the drug delivery system. Results indicated that the release of 5-Fu was controllable and the release half-time could reach as long as 54.46 h, much slower than that of free 5-Fu. Cytotoxicity evaluation and cellular apoptosis study suggested good biocompatibility of CS-g-PCL micelles. Moreover, 5-Fu loaded micelles could delay the release of drug and exert comparable cytotoxicity against A549 cells.

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.

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.

Post-transcriptional regulation of the tumor suppressor miR-139-5p and a network of miR-139-5p-mediated mRNA interactions in colorectal cancer.

MicroRNAs play key roles in many biological processes, and are frequently dysregulated in tumor cells. However, there are few studies on how microRNAs are dysregulated. miR-139-5p, an important tumor suppressor, is often underexpressed in gastrointestinal cancer cells. Here, we describe post-transcriptional regulation of this intronic microRNA in human colorectal cancer. miR-139-5p is expressed independently of its overexpressed host gene PDE2A in colorectal cancer tissues and cell lines. The miR-139-5p target genes IGF1R, ROCK2 and RAP1B exert regulatory effects on the miR-139-5p expression level, relying on their ability to compete for miR-139-5p binding. These overexpressed target genes also regulate each others' protein levels through 3'-UTRs, thus regulating tumor cell growth and motility properties. Our study provides a mechanistic, experimentally validated rationale for intronic microRNA dysregulation in colorectal cancer, revealing novel oncogenic roles of IGF1R, ROCK2 and RAP1B 3'-UTRs.

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.

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.