Investigation Of Novel Therapeutic Strategies Research Articles

CDK12 is a promising therapeutic target for the transcription cycle and DNA damage response in metastatic osteosarcoma.

Osteosarcoma (OS) is a bone malignant tumor affecting children, adolescents, and young adults. Currently, osteosarcoma is treated with chemotherapy regimens established over 40 years ago. The investigation of novel therapeutic strategies for the treatment of osteosarcoma remains an important clinical need. Cyclin-dependent kinases (CDKs) have been considered promising molecular targets in cancer therapy. Among these, CDK12 has been shown to play a crucial role in the pathogenesis of malignancies, but its clinical significance and biological mechanisms in osteosarcoma remain unclear. In the present study, we aim to determine the expression and function of CDK12 and evaluate its prognostic and therapeutic value in metastatic osteosarcoma. We found that overexpression of CDK12 was associated with high tumor grade, tumor progression and reduced patient survival. The underlying mechanism revealed that knockdown of CDK12 expression with small interfering RNA or functional inhibition with the CDK12-targeting agent THZ531 effectively exhibited time- and dose-dependent cytotoxicity. Downregulation of CDK12 paused transcription by reducing RNAP II phosphorylation, interfered with DNA damage repair with increased γH2AX, and decreased cell proliferation through the PI3K-AKT pathway. This was accompanied by the promotion of apoptosis, as evidenced by enhanced Bax expression and reduced Bcl-xL expression. Furthermore, the CDK12 selective inhibitor THZ531 also hindered ex vivo 3D spheroid formation, growth of in vitro 2D cell colony, and prevented cell mobility. Our findings highlight the clinical importance of CDK12 as a potentially valuable prognostic biomarker and therapeutic target in metastatic osteosarcoma.

Association of mental disorders with sepsis: a bidirectional Mendelian randomization study.

Substantial research evidence supports the correlation between mental disorders and sepsis. Nevertheless, the causal connection between a particular psychological disorder and sepsis remains unclear. For investigating the causal relationships between mental disorders and sepsis, genetic variants correlated with mental disorders, including anorexia nervosa (AN), attention-deficit hyperactivity disorder (ADHD), autism spectrum disorder (ASD), bipolar disorder (BD), major depressive disorder (MDD), obsessive-compulsive disorder (OCD), panic disorder (PD), posttraumatic stress disorder (PTSD), schizophrenia (SCZ), and tourette syndrome (TS), were all extracted from the Psychiatric Genomics Consortium (PGC). The causal estimates and direction between these mental disorders and sepsis were evaluated employing a two-sample bidirectional MR strategy. The inverse variance weighted (IVW) method was the primary approach utilized. Various sensitivity analyses were performed to confirm the validity of the causal effect. Meta-analysis, multivariable MR, and mediation MR were conducted to ensure the credibility and depth of this research. The presence of AN was in relation to a greater likelihood of sepsis (OR 1.08, 95% CI 1.02-1.14; p = 0.013). A meta-analysis including validation cohorts supported this observation (OR 1.06, 95% CI 1.02-1.09). None of the investigated mental disorders appeared to be impacted when sepsis was set as the exposure factor. Even after adjusting for confounding factors, AN remained statistically significant (OR 1.08, 95% CI 1.02-1.15; p = 0.013). Mediation analysis indicated N-formylmethionine levels (with a mediated proportion of 7.47%), cystatin D levels (2.97%), ketogluconate Metabolism (17.41%) and N10-formyl-tetrahydrofolate biosynthesis (20.06%) might serve as mediators in the pathogenesis of AN-sepsis. At the gene prediction level, two-sample bidirectional MR analysis revealed that mental disorder AN had a causal association with an increased likelihood of sepsis. In addition, N-formylmethionine levels, cystatin D levels, ketogluconate metabolism and N10-formyl-tetrahydrofolate biosynthesis may function as potential mediators in the pathophysiology of AN-sepsis. Our research may contribute to the investigation of novel therapeutic strategies for mental illness and sepsis.

The application of peroxidase mimetic nanozymes in cancer diagnosis and therapy

In recent decades, scholarly investigations have predominantly centered on nanomaterials possessing enzyme-like characteristics, commonly referred to as nanozymes. These nanozymes have emerged as viable substitutes for natural enzymes, offering simplicity, stability, and superior performance across various applications. Inorganic nanoparticles have been extensively employed in the emulation of enzymatic activity found in natural systems. Nanoparticles have shown a strong ability to mimic a number of enzyme-like functions. These systems have made a lot of progress thanks to the huge growth in nanotechnology research and the unique properties of nanomaterials. Our presentation will center on the kinetics, processes, and applications of peroxidase-like nanozymes. In this discourse, we will explore the various characteristics that exert an influence on the catalytic activity of nanozymes, with a particular emphasis on the prevailing problems and prospective consequences. This paper presents a thorough examination of the latest advancements achieved in the domain of peroxidase mimetic nanozymes in the context of cancer diagnosis and treatment. The primary focus is on their use in catalytic cancer therapy, alongside chemotherapy, phototherapy, sonodynamic therapy, radiation, and immunotherapy. The primary objective of this work is to offer theoretical and technical assistance for the prospective advancement of anticancer medications based on nanozymes. Moreover, it is anticipated that this will foster the investigation of novel therapeutic strategies aimed at achieving efficacious tumor therapy.

Molecular profiling of the stroke-induced alterations in the cerebral microvasculature reveals promising therapeutic candidates

Stroke-induced cerebral microvascular dysfunction contributes to aggravation of neuronal injury and compromises the efficacy of current reperfusion therapies. Understanding the molecular alterations in cerebral microvessels in stroke will provide original opportunities for scientific investigation of novel therapeutic strategies. Toward this goal, using a recently optimized method which minimizes cell activation and preserves endothelial cell interactions and RNA integrity, we conducted a genome-wide transcriptomic analysis of cerebral microvessels in a mouse model of stroke and compared these transcriptomic alterations with the ones observed in human, nonfatal, brain stroke lesions. Results from these unbiased comparative analyses have revealed the common alterations in mouse stroke microvessels and human stroke lesions and identified shared molecular features associated with vascular disease (e.g., Serpine1/Plasminogen Activator Inhibitor-1, Hemoxygenase-1), endothelial activation (e.g., Angiopoietin-2), and alterations in sphingolipid metabolism and signaling (e.g., Sphigosine-1-Phosphate Receptor 2). Sphingolipid profiling of mouse cerebral microvessels validated the transcript data and revealed the enrichment of sphingomyelin and sphingoid species in the cerebral microvasculature compared to brain and the stroke-induced increase in ceramide species. In summary, our study has identified novel molecular alterations in several microvessel-enriched, translationally relevant, and druggable targets, which are potent modulators of endothelial function. Our comparative analyses have revealed the presence of molecular features associated with cerebral microvascular dysfunction in human chronic stroke lesions. The results shared here provide a detailed resource for therapeutic discovery of candidates for neurovascular protection in stroke and potentially, other pathologies exhibiting cerebral microvascular dysfunction.

Precise cell therapy for liver fibrosis: Endothelial cell and macrophage therapy

Liver fibrosis is typically caused by chronic viral hepatitis and, more recently, fatty liver disease associated with obesity. There are currently no approved drugs for liver cirrhosis, and liver transplantation is limited by donor scarcity, thus driving the investigation of novel therapeutic strategies. The development of liver fibrosis presents with stage- and zone-dependent characteristics that manifest as distinct dynamic changes during vascularization and extracellular matrix (ECM) deposition. However, current cellular therapies do not consider the spatiotemporal variations of liver fibrosis without identifying the precise location and stage to administer the intervention to achieve optimal therapeutic effects. Herein, we focus on endothelial cell (EC) and macrophage therapy for liver fibrosis because of their important roles in regulating the spatiotemporal changes of vascularization and ECM deposition during liver fibrosis progression. Overall, this review summarizes the stage-dependent EC and macrophage therapy for liver fibrosis, elucidates their respective mechanisms, and exemplifies potential strategies to realize precise cell therapy by targeting specific liver zones.

Lippia sidoides and Lippia origanoides essential oils affect the viability, motility and ultrastructure of Trypanosoma cruzi

Chagas disease, caused by the protozoan Trypanosoma cruzi, is considered a public health problem. The current chemotherapy for this illness causes serious side effects and its use in the chronic phase of the disease is still controversial. In this regard, the investigation of novel therapeutic strategies remains a priority. The essential oils (EOs) from aromatic plants emerge as a promising source of bioactive compounds. In a previous work we reported the trypanocidal activity of the essential oils from the medicinal plants Lippia sidoides (LSEO) and Lippia origanoides (LOEO) against T. cruzi. Herein, we aimed to further investigate, in more details, the mode of action of LSEO and LOEO on the different developmental stages of this parasite. We showed that Lippia sidoides (LSEO) and Lippia origanoides (LOEO) induced a significant reduction in the percentage of macrophages infected by T. cruzi and in the number of intracellular parasites. Ultrastructural analysis showed that the treatment with both oils caused morphological changes consistent with loss of viability and cell death. The reduced staining with calcein and the increase in the proportion of HE–positive cells also demonstrated that LSEO and LOEO caused loss of parasite viability and membrane integrity. A considerable decrease in Rhodamine 123 and an increase in fluorescence intensity of MitoSox in LOEO were indicative of loss of mitochondrial potential and generation of reactive oxygen species, which ultimately lead to parasite death. Moreover, the optical tweezer analysis indicated that LOEO was more effective in reducing the motility of the epimastigotes. Taken together, our results demonstrated that the LSEO and LOEO are active against T. cruzi and constitute a promising drugs for the therapy of Chagas disease.

Downregulation of VEGF and upregulation of TL1A expression induce HUVEC apoptosis in response to high glucose stimuli.

High glucose‑induced endothelial cell apoptosis is considered to be the initiator of diabetes‑associated vascular complications. Experiments in vivo and in vitro have demonstrated that high glucose levels contribute to the apoptosis of endothelial cells by mediating cellular dysfunction and metabolic disorder via the production of various cytokines. As the most important endogenous vascular regulators, the balance between pro‑proliferative effector vascular endothelial growth factor (VEGF) and anti‑proliferative effector tumor necrosis factor‑like cytokine 1A (TL1A) is important in the modulation of endothelial cell survival and proliferation, and neovascularization. The present study aimed to explore whether the imbalance between VEGF and TL1A affected the apoptosis of human umbilical vein endothelial cells (HUVECs) exposed to high glucose conditions and then further investigated the potential mechanism. The results showed that the downregulation of VEGF in combination with the upregulation of TL1A in response to high glucose levels led to enhanced HUVEC apoptosis. Further experiments revealed that silencing high glucose‑induced TL1A expression using TL1A small interfering (si)RNA or the overexpression of VEGF by transfection with VEGF DNA resulted in a reduced HUVEC apoptosis rate compared with the controls. The effects occurred by attenuating and activating the phosphoinositide 3‑kinase/Akt/endothelial nitric oxide synthase pathway, respectively. In addition, VEGF and TL1A inhibited each other in hyperglycemia. In conclusion, these findings provide theoretical support for the further investigation of novel therapeutic strategies designed to maintain the balance between VEGF and TL1A and, thus, to prevent the onset and progression of endothelial cell apoptosis in response to high glucose stimuli.

New agents for targeting of IL-13RA2 expressed in primary human and canine brain tumors.

Interleukin 13 receptor alpha 2 (IL-13RA2) is over-expressed in a vast majority of human patients with high-grade astrocytomas like glioblastoma. Spontaneous astrocytomas in dogs resemble human disease and have been proposed as translational model system for investigation of novel therapeutic strategies for brain tumors. We have generated reagents for both detection and therapeutic targeting of IL-13RA2 in human and canine brain tumors. Peptides from three different regions of IL-13RA2 with 100% sequence identity between human and canine receptors were used as immunogens for generation of monoclonal antibodies. Recombinant canine mutant IL-13 (canIL-13.E13K) and canIL-13.E13K based cytotoxin were also produced. The antibodies were examined for their immunoreactivities in western blots, immunohistochemistry, immunofluorescence and cell binding assays using human and canine tumor specimen sections, tissue lysates and established cell lines; the cytotoxin was tested for specific cell killing. Several isolated MAbs were immunoreactive to IL-13RA2 in western blots of cell and tissue lysates from glioblastomas from both human and canine patients. Human and canine astrocytomas and oligodendrogliomas were also positive for IL-13RA2 to various degrees. Interestingly, both human and canine meningiomas also exhibited strong reactivity. Normal human and canine brain samples were virtually negative for IL-13RA2 using the newly generated MAbs. MAb 1E10B9 uniquely worked on tissue specimens and western blots, bound live cells and was internalized in GBM cells over-expressing IL-13RA2. The canIL-13.E13K cytotoxin was very potent and specific in killing canine GBM cell lines. Thus, we have obtained several monoclonal antibodies against IL-13RA2 cross-reacting with human and canine receptors. In addition to GBM, other brain tumors, such as high grade oligodendrogliomas, meningiomas and canine choroid plexus papillomas, appear to express the receptor at high levels and thus may be appropriate candidates for IL-13RA2-targeted imaging/therapies. Canine spontaneous primary brain tumors represent an excellent translational model for human counterparts.

Current status of Chagas disease chemotherapy

Chagas disease affects 7.7 million people and 28 million people are at risk of acquiring the disease in 15 endemic countries of Latin America. Benznidazole and nifurtimox are drugs that have been used to treat the disease. However, both drugs induce severe side effects. Treatment with benznidazole has been recommended for the acute phase (0–4 months after infection), recent chronic phase (children 0–14 years of age, treated 4 months after infection) and congenital infection. Average cure rates for Chagas disease patients obtained from clinical trials were 97.9% (congenital infection, treatment performed 0–6 months of age), 71.5% (acute phase), 57.6% (recent chronic phase, children 0–13 years of age) and 5.9% (late chronic phase, great majority of patients between 15 and 69 years of age). Clinical evidence about the capacity of antiparasitic treatment to avoid, stop or revert heart pathology in indeterminate and cardiac chronic patients is contradictory. The investigation of novel therapeutic strategies against Chagas disease remains a priority in the research of tropical diseases. Unfortunately, Chagas disease remains neglected in the formulation of strategies toward control of this disease. This article focuses on current therapeutic approaches to Chagas disease.

Cracking the system: melanoma complexity demands new therapeutic approaches

The melanoma field remains rich with discovery opportunities and stands poised for significant therapeutic advances. Although melanoma is notoriously resistant to chemotherapeutic and immunotherapeutic strategies, recent reports of complete clinical responses following the use of small molecule inhibitors in acral and mucosal metastatic melanomas harboring KIT alterations provide hope that melanomas can be managed clinically (Hodi et al., 2008). Given such significant patient responses, we have a hint that successful therapies do exist afterall against advanced melanoma and teasing out more positive outcomes should not be an impossible feat. ‘Magic bullets’ however, are not presently available for most melanomas and the poor efficacy of single-agent chemotherapeutic approaches warrants the investigation of novel therapeutic strategies and inhibitors. The path to target and drug discovery is now facilitated by increased access to powerful tools such as high-throughput sequencing machines, extensive compound libraries, and expanding panels of genetically diverse melanoma cell lines or samples. While a number of melanoma targets have already been flagged and await proper validation and inhibition in the clinical setting (mutant BRAF inhibitors come to mind), genespecific mutational analysis, and gene expression profiling are constantly exposing novel melanoma effectors and pathways that may prove just as valuable for melanoma research as those presently available, if not more. Using genomewide genetic analysis, Jones et al. (2008) most recently showed that 24 pancreatic cancers contain an average of 63 genetic alterations, which define a smaller key set of 12 signaling pathways and processes. In addition, these core pathways and processes are found altered in 67–100% of tumors analyzed. This novel and powerful approach to cancer research, which will be applied to melanoma shortly, is physiologically and therapeutically relevant given that it focuses the great genetic variations found across patients into fewer pathways that can be more easily studied and targeted. On a smaller scale, a study by Smalley et al. (2008a), showed that mutational analysis and use of a diverse panel of melanoma cell lines provide insights into melanoma resistance to BRAF inhibition, namely through the involvement of cyclin-dependent kinase 4 (CDK4) and ⁄ or cyclin D1 (CCND1). Unlike cell lines harboring a CDK4 mutation alone, cell lines with both a CDK4 mutation and CCND1 amplification were able to display resistance to BRAF inhibition. In addition, overexpression of cyclin D1 increased resistance to BRAF inhibition which was further potentiated by CDK4 overexpression. These findings hold clinical relevance since array comparative genomic hybridization analyses (aCGH) show CCND1 amplifications in 17% of BRAF V600E-mutated metastatic melanoma samples (Smalley et al., 2008a). CDK small molecule inhibitors are presently being developed and may prove powerful in combination with BRAF inhibitors. The observation that a great proportion of melanomas display constitutive mitogen-activated protein kinase (MAPK) pathway activity led to the eager development of a number of RAF and MEK inhibitors; however, these compounds in early clinical studies led to disappointing results despite encouraging preclinical findings. Current opinion now points to the need to target several pathways concomitantly in order to control melanoma growth, survival, and metastasis due to the presence of redundant protein functions and compensatory ⁄ adaptable signaling pathways. In fact, melanoma researchers are increasingly adopting therapeutic strategies from a new paradigm emerging in drug discovery: ‘network pharmacology’, which integrates systems biology, network analysis, connectivity, redundancy and pleiotropy. Such strategies include the use of rational multidrug combinations, exploiting the properties of approved drugs in combination, and using single compounds specifically binding to two or more diverse molecular targets in order to improve treatment efficacy (Hopkins, 2008). Exemplifying the use of the rational multidrug combination approach to potentiate a drug response, a recent study by Cragg et al. showed that MEK inhibition caused the upregulation of the proapoptotic Bcl-2 family member Bim in BRAF mutant melanoma and colon cancer cell lines. Given that apoptosis was minimal owing to the neutralizing effects of Bcl-2 and Bcl-xL, the Bcl-2 antagonist ABT-737 was employed causing the MEK inhibitioninduced cytostatic effect to shift to a cytotoxic effect with long-term tumor regression in a mouse xenograft model (Cragg et al., 2008). Coverage on: Jones et al. (2008). Core signaling pathways in human pancreatic cancers revealed by global genomic analyses. Science 321: 1801–1806. Smalley et al. (2008a). Increased cyclin D1 expression can mediate BRAF inhibitor resistance in BRAF V600E-mutated melanomas. Mol. Cancer Ther. 7: 2876– 2883. Cragg et al. (2008). Treatment of B-RAF mutant human tumor cells with a MEK inhibitor requires Bim and is enhanced by a BH3 mimetic. J. Clin. Invest. 118: 3651–3659. Smalley et al. (2008b). CRAF inhibition induces apoptosis in melanoma cells with non-V600E BRAF mutations. Oncogene [Epub ahead of print].

Murine and Canine Models of Appendicular Osteosarcoma

Appendicular osteosarcoma (OSA) is a primary bone sarcoma affecting humans during their second decade of life. Despite aggressive surgical and chemotherapeutic interventions, 30% of patients will experience progressive metastatic disease within 5 years of diagnosis. Understanding the biology of pediatric OSA and potential targets for therapeutic development remains an area of focus for both basic scientists and clinical oncologists. The identification and study of relevant comparative tumor models in mice and canines may allow for a better understanding of OSA biology, and permit the rapid investigation of novel therapeutic strategies for managing this metastatic bone sarcoma. This unit provides a protocol for using an orthotopic, syngeneic murine model of appendicular OSA as an investigative tool for the study of OSA biology. Additionally, the comparative relevance of spontaneously occurring appendicular OSA in canines for the study of pediatric bone sarcomas is discussed.

Second allogeneic hematopoietic stem cell transplantation in children to overcome graft failure or relapse after initial transplant

Purpose:Failure of hematopoietic stem cell transplantation(HSCT) may be encountered in practice because of either relapse of the malignancy or dysfunction of the graft. Second HSCT may be the only option for some patients whose initial HSCT failed. Methods:From May, 1991 to December, 2004, 115 HSCTs were performed at the Pediatric Blood & Marrow Transplantation Center, Chonnam National University. This study was a retrospective analysis of the medical records of 15 patients who received the second HSCT after initial graft. Results:Among eight patients with nonmalignant diseases, two patients underwent the second HSCT because of primary graft failure and five because of late graft rejection. The remaining Fanconi anemia patient was re-transplanted due to development of AML. Two patients died and one experienced primary graft failure, but is still alive. The Kaplan-Meier 5-year overall survival rate was 75 percent and the disease free survival rate was 62.5 percent in nonmalignant diseases. All malignant patients underwent second transplants because of relapses. Four died of relapse and one of treatment-related complications. The Kaplan-Meier 2-year overall and event free survival rate was 28.6 percent each in malignant diseases. Conclusion:Second HSCT for graft dysfunction of nonmalignant disease seems to be feasible and should be considered as a standard practice. The relapse of malignant diseases remains a big obstacle even after the second HSCT, although a small portion of patients might be salvaged. Further investigation of novel therapeutic strategies, as well an the understanding of the biology should be explored