Mechanisms and Αpplications of Ιnterleukins in Cancer Immunotherapy

Over the past years, advances in cancer immunotherapy have resulted in innovative and novel approaches in molecular cancer diagnostics and cancer therapeutic procedures. However, due to tumor heterogeneity and inter-tumoral discrepancy in tumor immunity, the clinical benefits are quite restricted. The goal of this review is to evaluate the major cytokines-interleukins involved in cancer immunotherapy and project their basic biochemical and clinical applications. Emphasis will be given to new cytokines in pre-clinical development, and potential directions for future investigation using cytokines. Furthermore, current interleukin-based approaches and clinical trial data from combination cancer immunotherapies will also be discussed. It appears that continuously increasing comprehension of cytokine-induced effects, cancer stemness, immunoediting, immune-surveillance as well as understanding of molecular interactions emerging in the tumor microenvironment and involving microRNAs, autophagy, epithelial-mesenchymal transition (EMT), inflammation, and DNA methylation processes may hold much promise in improving anti-tumor immunity. To this end, the emerging in-depth knowledge supports further studies on optimal synergistic combinations and additional adjuvant therapies to realize the full potential of cytokines as immunotherapeutic agents.     

Survivin as a Preferential Target for Cancer Therapy

Cancer is typically a consequence of imbalance between cell death and proliferation in a way favorable to cell proliferation and survival. Most conventional cancer therapies are based on targeting rapidly growing cancerous cells to block growth or enhance cell death, thereby, restoring the balance between these processes. In many instances, malignancies that develop resistance to current treatment modalities, such as chemotherapy, immunotherapy, and radiotherapy often present the greatest challenge in subsequent management of the patient. Studies have shown that under normal circumstances, cells utilize different death mechanisms, such as apoptosis (programmed cell death), autophagy, mitotic catastrophe, and necrosis to maintain homeostasis and physiological integrity of the organism, but these processes often appear to be altered in cancer. Thus, in recent years developing various strategies for administration of cytotoxic chemotherapeutics in combination with apoptosis-sensitizing reagents is receiving more emphasis. Here, we review the properties of the anti-apoptotic protein, survivin, a member of the inhibitor of apoptosis protein (IAP) family and the clinical feasibility and anti-cancer potential of drugs targeting this protein. We also discuss some key points and concerns that should be taken into consideration while developing drugs that target apoptotic proteins, such as survivin.     

Lipid Metabolism,Apoptosis and Cancer Therapy

Lipid metabolism is regulated by multiple signaling pathways, and generates a variety of bioactive lipid molecules. These bioactive lipid molecules known as signaling molecules, such as fatty acid, eicosanoids, diacylglycerol, phosphatidic acid, lysophophatidic acid, ceramide, sphingosine, sphingosine-1-phosphate, phosphatidylinositol-3 phosphate, and cholesterol, are involved in the activation or regulation of different signaling pathways. Lipid metabolism participates in the regulation of many cellular processes such as cell growth, proliferation, differentiation, survival, apoptosis, inflammation, motility, membrane homeostasis, chemotherapy response, and drug resistance. Bioactive lipid molecules promote apoptosis via the intrinsic pathway by modulating mitochondrial membrane permeability and activating different enzymes including caspases. In this review, we discuss recent data in the fields of lipid metabolism, lipid-mediated apoptosis, and cancer therapy. In conclusion, understanding the underlying molecular mechanism of lipid metabolism and the function of different lipid molecules could provide the basis for cancer cell death rationale, discover novel and potential targets, and develop new anticancer drugs for cancer therapy.     

Emerging Roles of Claudins in Human Cancer

Claudins are major integral membrane proteins of tight junctions. Altered expression of several claudin proteins, in particular claudin-1, -3, -4 and -7, has been linked to the development of various cancers. Although their dysregulation in cancer suggests that claudins play a role in tumorigenesis, the exact underlying mechanism remains unclear. The involvement of claudins in tumor progression was suggested by their important role in the migration, invasion and metastasis of cancer cells in a tissue-dependent manner. Recent studies have shown that they play a role in epithelial to mesenchymal transition (EMT), the formation of cancer stem cells or tumor-initiating cells (CSCs/TICs), and chemoresistance, suggesting that claudins are promising targets for the treatment of chemoresistant and recurrent tumors. A recently identified claudin-low breast cancer subtype that is characterized by the enrichment of EMT and stem cell-like features is significantly associated with disease recurrence, underscoring the importance of claudins as predictors of tumor recurrence. The critical role of epigenetic mechanisms in the regulation of claudin expression indicates the possible application of epigenetic therapy to target claudins. A better understanding of the emerging role of claudins in CSC/TICs and chemoresistance may help to develop therapies against recurrent cancers.     

Efficacy and Feasibility of the Epithelial Cell Adhesion Molecule (EpCAM) Immunomagnetic Cell Sorter for Studies of DNA Methylation in Colorectal Cancer

The aim of this work was to assess the impact on measurements of methylation of a panel of four cancer gene promoters of purifying tumor cells from colorectal tissue samples using the epithelial cell adhesion molecule (EpCAM)-immunomagnetic cell enrichment approach. We observed that, on average, methylation levels were higher in enriched cell fractions than in the whole tissue, but the difference was significant only for one out of four studied genes. In addition, there were strong correlations between methylation values for individual samples of whole tissue and the corresponding enriched cell fractions. Therefore, assays on whole tissue are likely to provide reliable estimates of tumor-specific methylation of cancer genes. However, tumor cell tissue separation using immunomagnetic beads could, in some cases, give a more accurate value of gene promoter methylation than the analysis of the whole cancer tissue, although relatively expensive and time-consuming. The efficacy and feasibility of the immunomagnetic cell sorting for methylation studies are discussed.     

NotI Microarrays: Novel Epigenetic Markers for Early Detection and Prognosis of High Grade Serous Ovarian Cancer

Chromosome 3-specific NotI microarray (NMA) containing 180 clones with 188 genes was used in the study to analyze 18 high grade serous ovarian cancer (HGSOC) samples and 7 benign ovarian tumors. We aimed to find novel methylation-dependent biomarkers for early detection and prognosis of HGSOC. Thirty five NotI markers showed frequency of methylation/deletion more or equal to 17%. To check the results of NMA hybridizations several samples for four genes (LRRC3B, THRB, ITGA9 and RBSP3 (CTDSPL)) were bisulfite sequenced and confirmed the results of NMA hybridization. A set of eight biomarkers: NKIRAS1/RPL15, THRB, RBPS3 (CTDSPL), IQSEC1, NBEAL2, ZIC4, LOC285205 and FOXP1, was identified as the most prominent set capable to detect both early and late stages of ovarian cancer. Sensitivity of this set is equal to (72 ± 11)% and specificity (94 ± 5)%. Early stages represented the most complicated cases for detection. To distinguish between Stages I + II and Stages III + IV of ovarian cancer the most perspective set of biomarkers would include LOC285205, CGGBP1, EPHB1 and NKIRAS1/RPL15. The sensitivity of the set is equal to (80 ± 13)% and the specificity is (88 ± 12)%. Using this technique we plan to validate this panel with new epithelial ovarian cancer samples and add markers from other chromosomes.     

Over-Expression of Platelet-Derived Growth Factor-D Promotes Tumor Growth and Invasion in Endometrial Cancer

The platelet-derived growth factor-D (PDGF-D) was demonstrated to be able to promote tumor growth and invasion in human malignancies. However, little is known about its roles in endometrial cancer. In the present study, we investigated the expression and functions of PDGF-D in human endometrial cancer. Alterations of PDGF-D mRNA and protein were determined by real time PCR, western blot and immunohistochemical staining. Up-regulation of PDGF-D was achieved by stably transfecting the pcDNA3-PDGF-D plasmids into ECC-1 cells; and knockdown of PDGF-D was achieved by transient transfection with siRNA-PDGF-D into Ishikawa cells. The MTT assay, colony formation assay and Transwell assay were used to detect the effects of PDGF-D on cellular proliferation and invasion. The xenograft assay was used to investigate the functions of PDGF-D in vivo. Compared to normal endometrium, more than 50% cancer samples showed over-expression of PDGF-D (p < 0.001), and high level of PDGF-D was correlated with late stage (p = 0.003), deep myometrium invasion (p < 0.001) and lympha vascular space invasion (p = 0.006). In vitro, over-expressing PDGF-D in ECC-1 cells significantly accelerated tumor growth and promoted cellular invasion by increasing the level of MMP2 and MMP9; while silencing PDGF-D in Ishikawa cells impaired cell proliferation and inhibited the invasion, through suppressing the expression of MMP2 and MMP9. Moreover, we also demonstrated that over-expressed PDGF-D could induce EMT and knockdown of PDGF-D blocked the EMT transition. Consistently, in xenografts assay, PDGF-D over-expression significantly promoted tumor growth and tumor weights. We demonstrated that PDGF-D was commonly over-expressed in endometrial cancer, which was associated with late stage deep myometrium invasion and lympha vascular space invasion. Both in vitro and in vivo experiments showed PDGF-D could promote tumor growth and invasion through up-regulating MMP2/9 and inducing EMT. Thus, we propose targeting PDGF-D to be a potent strategy for endometrial cancer treatment.     

人核糖核苷酸酶抑制因子表达下调对膀胱癌T24细胞在体内外发生上皮间质转化的影响

目的探讨人核糖核苷酸酶抑制因子(human ribonuclease inhibitor,hRI)表达下调对膀胱癌T24细胞在体内外发生上皮间质转化(epithelial-mesenchymal transition,EMT)的影响。方法干涉质粒pGensil-1-RI稳定转染T24细胞,经G418筛选阳性克隆,并设实验组、空质粒对照组及未转染对照组,RT-PCR检测各组细胞中RI基因mRNA的转录水平、细胞免疫荧光和Western blot检测RI蛋白的表达水平;HE染色检测细胞形态的变化;Western blot检测各组细胞中E-cadherin、Twist、Slug和Vimentin等相关蛋白表达水平;将3组细胞注入BALB/c裸鼠皮下,35 d后取瘤并称重;免疫组织化学检测瘤组织中RI、E-cadherin和Vimentin蛋白的表达水平。结果荧光显微镜下观察可见,干涉质粒pGensil-1-RI转染成功;实验组RI基因mRNA的转录水平和蛋白的表达水平较两对照组分别降低了63.31%和64.11%、49.6%和49.5%(P<0.001);实验组细胞呈梭形,核质比增大;与实验相比,两对照组E-cadhein蛋白表达水平分别增加52.76%和46.93%(P<0.001),而Twist、Slug和Vimentin蛋白分别降低了50.49%和54.63%、43.74%和51.55%、60.35%和53.77%(P<0.001);两对照组的瘤重明显低于实验组,分别低84.91%和80.89%(P<0.01),其RI和E-cadherin蛋白表达水平降低,而Vimentin蛋白表达水平则增加。结论沉默RI基因能明显增加T24细胞的转移、侵袭及EMT的能力。     

树突状细胞疫苗与宫颈癌细胞免疫治疗的新进展

宫颈癌是妇科最常见的恶性肿瘤之一,其发病率在女性恶性肿瘤中居第2位。树突状细胞(DC)是机体免疫应答的始动者,以DC为基础的抗肿瘤免疫治疗已成为热点。本文就树突状细胞的抗肿瘤机制及其在宫颈癌免疫治疗中的应用作一综述。     

骨桥蛋白反义基因对乳腺癌荷瘤裸鼠肿瘤生长及肺转移的影响

目的探讨骨桥蛋白反义基因(ANOPN)对乳腺癌荷瘤裸鼠肿瘤生长及肺转移的影响。方法构建人骨桥蛋白反义基因真核表达质粒pcDNA3.1-ANOPN,将质粒pcDNA3.1-ANOPN、空载体pcDNA3.1(+)和等量的脂质体分别转染人乳腺癌细胞系MDA-MA-231,将转染细胞分别命名为MDA-ANOPN、MDA-vect和MDA,接种至裸鼠,观察肿瘤的生长、转移情况,并用免疫组化法检测肿瘤组织切片中OPN的表达。结果重组质粒pcDNA3.1-ANOPN经酶切、PCR及测序,证明构建正确。MDA-ANOPN组裸鼠出瘤时间、肿瘤体积及重量较MDA-vect和MDA组均显著降低,肿瘤肺转移灶数量减少,OPN表达较低。结论ANOPN可抑制裸鼠移植瘤的生长及肺转移。     

Changes in Immunogenicity during the Development of Urinary Bladder Cancer: A Preliminary Study

In the present study, we evaluated tumor-infiltrating lymphocytes (TILs) and blood regulatory T lymphocyte (Tregs, CD4+/CD25+/FoxP3+) expression in bladder cancer patients. The number of CD4+, CD8+, CD25+, FoxP3+ and CD20+ TILs was analyzed in association with clinico-pathomorphological features. In more advanced metastasizing tumors, showing non-classic differentiation (ND) and a more aggressive tissue invasion type (TIT), the number of TILs decreased. A low number of CD4+ TILs was associated with poor prognosis. Similarly, Treg frequency before surgery and after surgical treatment was significantly lower in more advanced tumors. The changes in TILs, as well as of local and systemic Tregs, were accompanied by changes in the histological phenotype of urothelial carcinoma regarding pT stage, NDs, TIT, and clinical outcomes. The number of TILs and the frequency of blood Tregs (indicators of antitumor response) may be essential for choosing an immunotherapy that is adjusted to the immune status according to the phase of tumor growth. Moreover, a significant reduction in the number of CD4+ and CD8+ TILs with the development of NDs in more advanced tumors may be associated with lower tumor immunogenicity, resulting in immune tolerance towards tumor tissue. These observations and the tendency of urothelial bladder carcinoma to undergo NDs in a heterogeneous manner during tumor progression suggest complex interactions between bladder cancer immunogenicity and stages of tumor progression.     

Multistep Model of Cervical Cancer: Participation of miRNAs and Coding Genes

Aberrant miRNA expression is well recognized as an important step in the development of cancer. Close to 70 microRNAs (miRNAs) have been implicated in cervical cancer up to now, nevertheless it is unknown if aberrant miRNA expression causes the onset of cervical cancer. One of the best ways to address this issue is through a multistep model of carcinogenesis. In the progression of cervical cancer there are three well-established steps to reach cancer that we used in the model proposed here. The first step of the model comprises the gene changes that occur in normal cells to be transformed into immortal cells (CIN 1), the second comprises immortal cell changes to tumorigenic cells (CIN 2), the third step includes cell changes to increase tumorigenic capacity (CIN 3), and the final step covers tumorigenic changes to carcinogenic cells. Altered miRNAs and their target genes are located in each one of the four steps of the multistep model of carcinogenesis. miRNA expression has shown discrepancies in different works; therefore, in this model we include miRNAs recording similar results in at least two studies. The present model is a useful insight into studying potential prognostic, diagnostic, and therapeutic miRNAs.     

Bioactive Carbohydrates and Peptides in Foods: An Overview of Sources,Downstream Processing Steps and Associated Bioactivities

Bioactive peptides and carbohydrates are sourced from a myriad of plant, animal and insects and have huge potential for use as food ingredients and pharmaceuticals. However, downstream processing bottlenecks hinder the potential use of these natural bioactive compounds and add cost to production processes. This review discusses the health benefits and bioactivities associated with peptides and carbohydrates of natural origin and downstream processing methodologies and novel processes which may be used to overcome these.