Altered Signal Transduction Research Articles

Prenatal Methamphetamine Hydrochloride Exposure Leads to Signal Transduction Alteration and Cell Death in the Prefrontal Cortex and Amygdala of Male and Female Rats' Offspring.

In the last decade, there has been a great increase in methamphetamine hydrochloride (METH) abuse by pregnant women that exposes fetus and human offspring to a wide variety of developmental impairments that may be the underlying causes of future psychosocial issues. Herein, we investigated whether prenatal METH exposure with different doses (2 and 5mg/kg) could influence neuronal cell death and antioxidant level in the different brain regions of adult male and female offspring. Adult male and female Wistar rats prenatally exposed to METH (2 or 5mg/kg) and/or saline was used in this study. At week 12, adult rats' offspring were decapitated to collect different brain region tissues including amygdala (AMY) and prefrontal cortices (PFC). Western blot analysis was performed to evaluate the apoptosis- and autophagy-related markers, and enzymatic assay was used to measure the level of catalase and also reduced glutathione (GSH). Our results showed that METH exposure during pregnancy increased the level of apoptosis (BAX/Bcl-2 and Caspase-3) and autophagy (Beclin-1 and LC3II/LC3I) in the PFC and AMY areas of both male and female offspring's brain. Also, we found an elevation in the GSH content of all both mentioned brain areas and catalase activity of PFC in the offspring's brain. These changes were more significant in female offspring. Being prenatally exposed to METH increased cell death at least partly via apoptosis and autophagy in AMY and PFC of male and female offspring's brain, while the antioxidant system tried to protect cells in these regions.

Integrated physiological and transcriptomic analyses reveal the molecular mechanism behind the response to cultivation in Quercus mongolica.

Quercus mongolica, a common tree species for building and landscaping in northern China, has great commercial and ecological value. The seedlings of Q. mongolica grow poorly and develop chlorosis when introduced from high-altitude mountains to low-altitude plains. Effective cultivation measures are key to improving the quality of seedlings. To investigate the complex responses of Q. mongolica to different cultivation measures, we compared the adaptability of 3-year-old Q. mongolica seedlings to pruning (P), irrigation (W), and fertilization [F (nitro compound fertilizer with 16N-16P-16K)]. Physiological measurements and transcriptome sequencing were performed on leaves collected under the P treatments (control, cutting, removal of all lateral branches, and removal of base branches to one-third of seedling height), the W treatments (0, 1, 2, 3, 4, or 5 times in sequence), and the F treatments (0, 2, 4, and 6 g/plant). Analyses of the physiological data showed that P was more effective than W or F for activating intracellular antioxidant systems. By contrast, W and F were more beneficial than P for inducing the accumulation of soluble sugar. OPLS-DA identified superoxide dismutase, malondialdehyde, and peroxidase as critical physiological indices for the three cultivation measures. Transcriptome analyses revealed 1,012 differentially expressed genes (DEGs) in the P treatment, 1,035 DEGs in the W treatment, and 1,175 DEGs in the F treatment; these DEGs were mainly enriched in Gene Ontology terms related to the stress response and signal transduction. Weighted gene coexpression network analyses indicated that specific gene modules were significantly correlated with MDA (one module) and soluble sugar (four modules). Functional annotation of the hub genes differentially expressed in MDA and soluble sugar-related modules revealed that Q. mongolica responded and adapted to different cultivation measures by altering signal transduction, hormone levels, reactive oxygen species, metabolism, and transcription factors. The hub genes HOP3, CIPK11, WRKY22, and BHLH35 in the coexpression networks may played a central role in responses to the cultivation practices. These results reveal the mechanism behind the response of Q. mongolica to different cultivation measures at the physiological and molecular levels and provide insight into the response of plants to cultivation measures.

Exposure to iprodione induces ROS production and mitochondrial dysfunction in porcine trophectoderm and uterine luminal epithelial cells, leading to implantation defects during early pregnancy

Iprodione is a well-known fungicide used in the cultivation of strawberries, tomatoes, grapes, and green beans. In recent studies, neurotoxicity, cardiotoxicity, and endocrine toxicity of iprodione have been reported. Although reproductive toxicity of iprodione has been identified in animal studies, its effects are limited to male fertility. Also, the toxic effects of iprodione on pregnancy, especially the implantation process, have not been elucidated. This study demonstrated a series of cytotoxic responses of iprodione along with the alteration of implantation-related gene expression in porcine trophectoderm (pTr) and luminal epithelium (pLE) cells. In this study, iprodione suppressed cell viability, proliferation, and migration of these cells. Iprodione induced G1 phase arrest and attenuated spheroid formation by pTr and pLE cells. Furthermore, iprodione caused mitochondrial dysfunction and excessive reactive oxygen species generation, which resulted in an increase in mitochondrial calcium levels. Consequently, DNA damage and apoptotic cell death were induced by iprodione treatment in pTr and pLE cells. This stress-induced cell death was mediated by alterations in intracellular signal transduction, including the PI3K/AKT and MAPK signaling pathways. This finding suggests the potential of iprodione to impair the implantation capacity by exerting cytotoxic effects on fetal and maternal cells.

Class I PI3K regulatory subunits control differentiation of dendritic cell subsets and regulate Flt3L mediated signal transduction

Dendritic cells (DCs) play pivotal roles in initiating and shaping both innate and adaptive immune responses. The spatiotemporal expression of transcription factor networks and activation of specific signal transduction pathways determine the specification, distribution and differentiation of DC subsets. Even though pioneering studies have established indispensable roles for specific catalytic subunits (p110δ and p110γ) in immune cells, functions of the regulatory subunits, particularly of Class I PI3K, within the hematopoietic system remain incompletely understood. In the study presented here, we deleted the key regulatory subunits—p85α and p85β of the Class IA PI3K in hematopoietic cells and studied its impact on DC differentiation. Our studies identify that a deficiency of p85 causes increased differentiation of conventional DC (cDC) 2 and plasmacytoid DC (pDC) subsets in the spleen. On the other hand, DC numbers in the bone marrow (BM), thymus and lymph nodes were decreased in p85 mutant mice. Analysis of DC-specific progenitors and precursors indicated increased numbers in the BM and spleen of p85 deficient mice. In-vitro differentiation studies demonstrated augmented DC-differentiation capacities of p85 deficient BM cells in the presence of GM-CSF and Flt3L. BM chimera studies established that p85 deficiency affects DC development through cell intrinsic mechanisms. Molecular studies revealed increased proliferation of DCs and common DC progenitors (CDPs) in the absence of p85 and altered signal transduction pathways in p85 mutant DC subsets in response to Flt3L. In essence, data presented here, for the first time, unequivocally establish that the P85α subunit of class IA PI3Ks has an indispensable role in the development and maintenance of DCs.

Oxidative Stress: Meeting Multiple Targets in Pathogenesis of Vascular Endothelial Dysfunction

The vascular endothelium is the innermost lining of blood vessels, which maintains vasoconstriction and vasodilation. Loss of vascular tone is a hallmark for cardiovascular disorders. Numerous factors, such as over-activation of the renin-angiotensin-aldosterone system, kinases, growth factors, etc., play a crucial role in the induction and progression of vascular abrasion. Interestingly, dysregulation of these pathways either enhances the intensity of oxidative stress, or these pathways are affected by oxidative stress. Thus, oxidative stress has been considered a key culprit in the progression of vascular endothelial dysfunction. Oxidative stress induced by reactive oxygen and nitrogen species causes abnormal gene expression, alteration in signal transduction, and the activation of pathways, leading to induction and progression of vascular injury. In addition, numerous antioxidants have been noted to possess promising therapeutic potential in preventing the development of vascular endothelial dysfunction. Therefore, we have focused on current perspectives in oxidative stress signalling to evaluate common biological processes whereby oxidative stress plays a crucial role in the progression of vascular endothelial dysfunction.

Abstract 2009: Elucidating the mechanistic effect of targeting Ref-1 redox function on MPNST survival signaling using patient-derived xenolines

Abstract Malignant Peripheral Nerve Sheath Tumor (MPNST) is a rare soft tissue sarcoma common in patients with NF1 (neurofibromatosis type 1). MPNSTs respond poorly to most chemotherapeutics due to molecular heterogeneity and altered signal transduction pathways. Ref-1 and STAT3 are highly expressed in MPNST patient samples offering druggable pathways. Inhibition of one singular protein, like Ref-1 to block the activity of many important transcription factors (TFs), STAT3, HIF1a, and NFkB is key to improving success in MPNST therapy. Inhibition of both Ref-1 and STAT3 in MPNST lines resulted in decreased proliferation, wound healing, tumor signaling, and deactivation of MPNST survival genes. Further, knockdown of Ref-1 or STAT3 resulted in a concordant decrease in NFkB activity. Ref-1 redox inhibitor, APX3330 that completed Phase I clinical trial (NCT03375086), potently inhibited in vitro growth of a panel of MPNST cells. We have also been developing new more potent analogs of APX3330 for inhibition of Ref-1 redox function and potent cell killing in our panel of MPNST cells. Several of these analogs significantly and potently reduced NFkB and HIF1a activity at concentrations where cell killing was minimal, pointing toward an on-target effect. Based on the role of Ref-1 in transcriptional regulation of MPNST, RNA sequencing after knockdown of Ref-1 was used to determine mechanistic effects on MPNST gene expression. We have identified 443 genes up-regulated and 758 genes down-regulated in two MPNST cell lines with siRef-1. The pathways enriched by the commonly up-regulated genes included RNA polymerase, P53 downstream, glycerophospholipid, and other lipid metabolism pathways; the pathways enriched by the commonly down-regulated genes included cell cycle, adaptive immune response, and VEGF signaling pathways. From this data, we also found that OXPHOS (Oxidative Phosphorylation) pathway genes (like NDUFS2, SURF1, COX15) were down with siRef-1 along with others like AURKA, RNASEH2A, CDC20, GINS4, TIMELESS that were identified in our previous publication to be MPNST survival genes. Based on our published observations that Ref-1 inhibition dramatically affects metabolic pathways, we used OXPHOS deficient and proficient osteosarcoma cells and confirmed the impact of Ref-1 redox activity on metabolism. Furthermore, if we combine Ref-1 inhibition with a-ketoglutarate (aKG) and target the tumor cells’ dependence on aspartate biosynthesis, the tumor cell death was dramatic (p < 0.0001). Two new xenolines were established from patient PDXs and are being validated for growth inhibition and downregulation of MPNST survival genes with Ref-1 knockdown and redox inhibition using APX analogs both in vitro and in vivo. Successful derailing of MPNST survival pathways by targeting Ref-1 redox function is our aim to treat this rare but deadly cancer. Citation Format: Silpa Gampala, Olivia Babb, Nikkitha Umesh Ganesh, Steven D. Rhodes, Reza M. Saadatzadeh, Kai Pollard, Christine Pratilas, Jing-Ruey Joanna Yeh, Karen E. Pollok, Wade D. Clapp, Mark R. Kelley, Chi Zhang, Melissa L. Fishel. Elucidating the mechanistic effect of targeting Ref-1 redox function on MPNST survival signaling using patient-derived xenolines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2009.

A reengineered common chain cytokine augments CD8+ T cell-dependent immunotherapy.

Cytokine therapy is limited by undesirable off-target side effects as well as terminal differentiation and exhaustion of chronically stimulated T cells. Here, we describe the signaling properties of a potentially unique cytokine by design, where T cell surface binding and signaling are separated between 2 different families of receptors. This fusion protein cytokine, called OMCPmutIL-2, bound with high affinity to the cytotoxic lymphocyte-defining immunoreceptor NKG2D but signaled through the common γ chain cytokine receptor. In addition to precise activation of cytotoxic T cells due to redirected binding, OMCPmutIL-2 resulted in superior activation of both human and murine CD8+ T cells by improving their survival and memory cell generation and decreasing exhaustion. This functional improvement was the direct result of altered signal transduction based on the reorganization of surface membrane lipid rafts that led to Janus kinase-3–mediated phosphorylation of the T cell receptor rather than STAT/AKT signaling intermediates. This potentially novel signaling pathway increased CD8+ T cell response to low-affinity antigens, activated nuclear factor of activated T cells transcription factors, and promoted mitochondrial biogenesis. OMCPmutIL-2 thus outperformed other common γ chain cytokines as a catalyst for in vitro CD8+ T cell expansion and in vivo CD8+ T cell–based immunotherapy.

ST6Gal1 Contributes to Pancreatic Cancer Initiation by Promoting Pancreatitis‐Induced Acinar to Ductal Metaplasia

Aberrant glycosylation is one of the earliest known hallmarks of cancer. Amongst the predominate changes in tumor cell glycosylation is an increase in α2‐6 sialylation on N‐glycans, a modification elaborated by the ST6Gal1 sialyltransferase. ST6Gal1 activity dramatically alters cellular function via the sialylation of various surface receptors such as EGFR and TNFR1, resulting in altered signal transduction and gene expression. ST6Gal1 expression is upregulated in multiple cancers, including pancreatic ductal adenocarcinoma (PDAC). Recent studies from our group suggest that the upregulation of ST6Gal1 in pancreatic acinar cells may promote PDAC initiation by facilitating acinar to ductal metaplasia (ADM), a process in which acinar cells de‐differentiate into ductal‐like, progenitor cells. In the nonmalignant pancreas, ADM is induced primarily by pancreatitis, a known risk factor for PDAC. Pancreatitis causes acinar cell apoptosis, however some cells undergo ADM and acquire regenerative capabilities essential for tissue healing. Notably, ADM‐like cells are particularly susceptible to oncogenic transformation. In the current study we determined that ST6Gal1 was strikingly upregulated in pancreatic tissues from chronic pancreatitis patients and mice with experimental pancreatitis. Furthermore, ST6Gal1 was selectively expressed in the ADM‐like cells, as identified both morphologically and by staining for ADM‐specific markers. ST6Gal1 activity was associated with a proliferative phenotype. More than 50% of cells with upregulated ST6Gal1 co‐expressed the proliferative marker, Ki67, whereas activated caspase 8 was undetectable in ST6Gal1‐positive cells. To interrogate a functional role for ST6Gal1 in ADM, we developed genetically‐engineered mice with ST6Gal1 expression in the pancreas (“SC” mice). Importantly, acinar cells from SC mice exhibited ADM‐like characteristics, indicated by increased expression of classic stem and ductal genes. RNASeq analyses comparing SC and WT pancreata indicated that SC cells had an enrichment in: stem cell pathways; a pancreatic ductal cell program; and gene networks associated with pancreatic cancer. Additionally, organoids derived from SC pancreata displayed increased expression of stem and ductal genes relative to WT organoids, as well as greater organoid‐forming potential and growth. To evaluate inflammation‐induced ADM, SC and WT mice were injected with cerulein to induce pancreatitis, and the ADM‐like cells were quantified by examining surface ADM markers by flow cytometry. Following induction of pancreatitis, more ADM‐like cells were present in SC versus WT pancreata. Finally, we crossbred SC mice to the “KC” PDAC model, which expresses oncogenic KRas (KrasG12D) in the pancreas. Mice with dual expression of ST6Gal1 and KrasG12D had greatly accelerated PDAC initiation, progression and mortality as compared with KC mice. Together these data suggest that the upregulation of ST6Gal1 during chronic pancreatitis may prime acinar cells for neoplastic transformation by facilitating ADM.

Chimeric Cytokine Targeting NKG2D and IL-2 Receptor Augments CD8+ T Cell-Metabolic Fitness and Improves Immunotherapy

Abstract T cell mediated immunotherapy has gained credible traction for cancer treatment. To date experimental and clinical strategies have relied on antibody-mediated co-stimulatory blockade, or the administration of high doses of naturally occurring or engineered cytokines to increase T cell activation. Cytokine therapy is by limited undesirable off-target side effects as well as terminal differentiation and exhaustion of chronically stimulated T cells. Here we describe functionality of a unique chimeric cytokine by design, called OMCPmutIL-2, binds with high affinity to the cytotoxic lymphocyte-defining immunoreceptor NKG2D but signals through the common γ-chain cytokine receptor, thus eliminating IL-2Ra mediated off-target side effects. In addition to precise activation of cytotoxic T cells due to redirected binding we describe that OMCPmutIL-2 results in superior activation of both human and murine CD8+ T cells by improving their survival, decreasing exhaustion, and improving memory cell generation. This functional improvement is the result altered signal transduction based on the reorganization of surface membrane lipid rafts resulting in JAK-mediated phosphorylation of the T cell receptor (TCR) rather than canonical STAT/AKT signaling intermediates activated by other common γ-chain cytokines. This altered signaling pathway increases CD8+ T cell response to low affinity antigens, activates Nuclear Factor of Activated T Cells (NFAT) transcription factors, and enhances mitochondrial biogenesis with improved T cell survival, cytotoxicity, and memory cell generation. OMCPmutIL-2 thus outperforms other common γ-chain cytokines as both a reagent for T cell expansion and in vivo CD8+ T cell-based immunotherapy. Supported by grant I01 BX002299

3D cell culture alters signal transduction and drug response in head and neck squamous cell carcinoma.

Epidermal growth factor receptor (EGFR) upregulation is a typical characteristic of head and neck squamous cell carcinoma (HNSCC). However, tyrosine kinase inhibitors have not yet been able to achieve enough therapeutic benefit in clinical trials to justify their use in standard therapy regimens. At present, little is known about the reasons for this treatment failure. In the present study, the HNSCC cell lines UM-SCC-11B and UM-SCC-22B were tested for their response to tyrosine kinase inhibitors (TKI) under 2D and 3D cell culture conditions. Absorption and luciferase-based viability assays were used for this, as well as optical evaluation via fluorescence microscopy. In addition, EGFR and HER3 expression as well as the downstream signalling pathways PI3K/AKT/mTOR and RAS/RAF/MEK/ERK were investigated using western blotting. Cell line UM-SCC-11B revealed a strong resistance to lapatinib under 3D cell culture conditions, while a good response to TKI therapy was observed under 2D cell culture conditions. An associated overexpression of phosphorylated HER3 under 3D cell culture conditions offered a plausible explanation for the altered treatment response. The results of the present study represent an idea of how signalling mechanisms of cancer cells can be changed using different cell culture methods. Overall, 3D cell culture could be an important component in the analysis of resistance mechanisms in cancer therapy.

Decreased Ankyrin Expression Is Associated with Repressed eNOS Signaling, Cell Proliferation, and Osteogenic Differentiation in Osteonecrosis of the Femoral Head.

Reduced nitric oxide synthase (NOS) activity and decreased reparative potentials in stem cells may be involved in the pathogenesis of osteonecrosis of the femoral head (ONFH), but the underlying mechanism is not clear. Ankyrin, a cytoskeletal protein, can promote NOS expression and many cellular functions when it interacts with the CD44 receptors on the stem cells. This study investigated whether ankyrin is involved in the pathogenesis of ONFH. Bone marrow stem cells (BMSCs) from ONFH patients were compared with cells from patients with proximal femoral fracture and BMSC cell lines (PT-2501, Lonza, NC, USA). Differences in the expression levels and downstream signal pathway of ankyrin-Akt-eNOS in BMSCs were studied between ONFH and control. The involvement of ankyrin in the signal cascade, cell proliferation, and differentiation were further investigated by silencing ankyrin using small interfering (si)RNA. We found the basal mRNA levels of ankyrin and CD44 in BMSCs from the ONFH group were significantly lower as compared with those from the control group. The signal transduction of CD44-ankyrin-Akt-eNOS was significantly repressed in the ONFH group as compared with the control group after hyaluronic acid treatment. Knockdown of ankyrin by siRNA could attenuate the eNOS signaling as well as the BMSCs proliferation and osteogenic differentiation. The proliferation ability and osteogenic differentiation potential of the BMSCs from the ONFH group were significantly reduced as compared with the control group, but they can be enhanced to the baseline levels of the control group by hyaluronic acid treatment. The aberrant eNOS signaling, reduced cell proliferation, and osteogenic differentiation potential in BMSCs from ONFH patients are associated with the decreased ankyrin expression. Altered signal transduction, proliferation, and osteogenic differentiation ability in BMSCs may be involved in the pathogenesis of ONFH. These need further studies especially in BMSC-based cell therapy.

Whole genome re-sequencing and transcriptome reveal an alteration in hormone signal transduction in a more-branching mutant of apple.

Branch number is an important trait in grafted apple breeding and cultivation. To provide new information on molecular mechanisms of apple branching, whole reduced-representation genomes and transcriptome of a wild-type (WT) apple (Malus spectabilis) and its more-branching (MB) mutant at the branching stage were examined in this study. Comparison of WT and MB genomes against the Malus domestica reference genome identified 14,908,939 single nucleotide polymorphisms (SNPs) and 173,315 insertions and deletions (InDels) in WT and 1,483,221 SNPs and 1,725,977 InDels in MB. Analysis of the genetic variation between MB and WT revealed 1,048,575 SNPs and 37,327 InDels. Among them, 24,303 SNPs and 891 InDels mapped to coding regions of 5,072 and 596 genes, respectively. GO and KEGG functional annotation of 3,846 and 944 genes, respectively, identified 32 variant genes related to plant hormone signal transduction that were involved in auxin, cytokinin, gibberellin, abscisic acid, ethylene, and brassinosteroid pathways. The transcriptome pathways of plant hormone signal transduction and zeatin biosynthesis were also significantly enriched during MB branching. Furthermore, transcriptome data suggested the regulatory roles of auxin signaling, increase of cytokinin and genes of cytokinin synthesis and signaling, and the suppressed abscisic acid signaling. Our findings suggest that branching development in apple is regulated by plant hormone signal transduction.

Inhibition of Musashi-1 enhances chemotherapeutic sensitivity in gastric cancer patient-derived xenografts.

Musashi-1 (MSI1), a neural RNA-binding protein, is considered a gastric and intestinal stem cell marker. Although the function of MSI1 in gastric cancer has attracted increasing interest, it is not known whether MSI1 can be used as a biomarker to monitor gastric cancer development and response to treatment. Here, the role of MSI1 in the chemotherapeutic sensitivity of gastric cancer was investigated. Patients with high MSI1 levels had poor outcomes, implicating the gene in the development and progression of the disease. We overexpressed and silenced MSI1 in the human gastric cancer cell lines MKN45 and HGC27, finding that knockdown reduced proliferation, invasion, and migration, while promoting apoptosis. A patient-derived xenograft gastric cancer model was constructed in which mice received chemical drugs, si-MSI1, or a drug-si-MSI1 combination. It was found that blocking MSI1 expression reduced gastric cancer drug tolerance. The combination treatment with si-MSI1 was superior to 5F-dUMP and cisplatin, either separately or in combination, indicating that including si-MSI1 was better than drug therapy alone. Transcriptome sequencing analysis showed that MSI1 altered cell cycle regulation and growth signal transduction, including that of blood vessel epicardial substance (BVES). These results suggest that MSI1 reduces the tolerance of gastric cancer to chemical drugs through modulation of MSI1/BVES signaling.

Effect of Environmental Stressors, Xenobiotics, and Oxidative Stress on Male Reproductive and Sexual Health.

This article examines the environmental factor-induced oxidative stress (OS) and their effects on male reproductive and sexual health. There are several factors that induce OS, i.e. radition, metal contamination, xenobiotic compounds, and cigarette smoke and lead to cause toxicity in the cells through metabolic or bioenergetic processes. These environmental factors may produce free radicals and enhance the reactive oxygen species (ROS). Free radicals are molecules that include oxygen and disbalance the amount of electrons that can create major chemical chains in the body and cause oxidation. Oxidative damage to cells may impair male fertility and lead to abnormal embryonic development. Moreover, it does not only cause a vast number of health issues such as ageing, cancer, atherosclerosis, insulin resistance, diabetes mellitus, cardiovascular diseases, ischemia-reperfusion injury, and neurodegenerative disorders but also decreases the motility of spermatozoa while increasing sperm DNA damage, impairing sperm mitochondrial membrane lipids and protein kinases. This chapter mainly focuses on the environmental stressors with further discussion on the mechanisms causing congenital impairments due to poor sexual health and transmitting altered signal transduction pathways in male gonadal tissues.

Biomedical Applications of Non-Small Cell Lung Cancer Spheroids.

Lung malignancies accounted for 11% of cancers worldwide in 2020 and remained the leading cause of cancer deaths. About 80% of lung cancers belong to non-small cell lung cancer (NSCLC), which is characterized by extremely high clonal and morphological heterogeneity of tumors and development of multidrug resistance. The improvement of current therapeutic strategies includes several directions. First, increasing knowledge in cancer biology results in better understanding of the mechanisms underlying malignant transformation, alterations in signal transduction, and crosstalk between cancer cells and the tumor microenvironment, including immune cells. In turn, it leads to the discovery of important molecular targets in cancer development, which might be affected pharmaceutically. The second direction focuses on the screening of novel drug candidates, synthetic or from natural sources. Finally, “personalization” of a therapeutic strategy enables maximal damage to the tumor of a patient. The personalization of treatment can be based on the drug screening performed using patient-derived tumor xenografts or in vitro patient-derived cell models. 3D multicellular cancer spheroids, generated from cancer cell lines or tumor-isolated cells, seem to be a helpful tool for the improvement of current NSCLC therapies. Spheroids are used as a tumor-mimicking in vitro model for screening of novel drugs, analysis of intercellular interactions, and oncogenic cell signaling. Moreover, several studies with tumor-derived spheroids suggest this model for the choice of “personalized” therapy. Here we aim to give an overview of the different applications of NSCLC spheroids and discuss the potential contribution of the spheroid model to the development of anticancer strategies.

Fibronectin: Molecular Structure, Fibrillar Structure and Mechanochemical Signaling.

The extracellular matrix (ECM) plays a key role as both structural scaffold and regulator of cell signal transduction in tissues. In times of ECM assembly and turnover, cells upregulate assembly of the ECM protein, fibronectin (FN). FN is assembled by cells into viscoelastic fibrils that can bind upward of 40 distinct growth factors and cytokines. These fibrils play a key role in assembling a provisional ECM during embryonic development and wound healing. Fibril assembly is also often upregulated during disease states, including cancer and fibrotic diseases. FN fibrils have unique mechanical properties, which allow them to alter mechanotransduction signals sensed and relayed by cells. Binding of soluble growth factors to FN fibrils alters signal transduction from these proteins, while binding of other ECM proteins, including collagens, elastins, and proteoglycans, to FN fibrils facilitates the maturation and tissue specificity of the ECM. In this review, we will discuss the assembly of FN fibrils from individual FN molecules; the composition, structure, and mechanics of FN fibrils; the interaction of FN fibrils with other ECM proteins and growth factors; the role of FN in transmitting mechanobiology signaling events; and approaches for studying the mechanics of FN fibrils.

Neuropathological Effects of Chemotherapeutic Drugs.

Novel treatments, screening, and detection methods have prolonged the lives of numerous cancer patients worldwide. Unfortunately, existing and many promising new chemotherapeutics can cause deleterious, off-target side effects in normal tissue and organ systems. The central and peripheral nervous systems are widely recognized as frequent off-target effectors of anticancer drugs which can produce persistent neurological and neuropsychiatric symptoms collectively termed "chemobrain". Following chemotherapy, patients report several forms of cognitive impairment occurring acutely and sometimes persisting years after treatment. There are no effective treatments for cognitive decline induced by chemotherapeutics, and the underlying molecular mechanisms are poorly characterized and understood. In this study, we find that chronic treatment with two common chemotherapeutic agents, cisplatin and gemcitabine, impairs brain region-specific metabolism, hippocampus-dependent memory formation, and stress response behavior. This corresponds to reduced hippocampal synaptic excitability, altered neuronal signal transduction, and neuroinflammation. These findings underline that a better understanding of the basic pathological consequences of chemotherapy-induced cognitive impairment is the first step toward improving cancer treatment survivorship.

Targeting molecular signal transduction pathways in hepatocellular carcinoma and its implications for cancer therapy.

Hepatocellular carcinoma is a substantial health concern. It is currently the third dominating cause of mortality associated with cancer worldwide. The development of hepatocellular carcinoma is an intricate process that encompasses the impairment of genetic, epigenetic, and signal transduction mechanisms contributing to an aberrant metabolic system, enabling tumorigenesis. Throughout the past decade, research has led to the revelation of molecular pathways implicated in the progression of this notorious disorder. The altered signal transduction pathways, such as the mitogen-activated protein kinase pathway, phosphoinositol 3-kinase/protein kinase B/mammalian target of rapamycin pathway, WNT/β-catenin pathway, hepatocyte growth factor/c-MET pathway, and just another kinase/signal transducers and activators of transcription signaling pathway is of much therapeutic significance, as targeting them may avail to revert, retard or avert hepatocarcinogenesis. The present review article sums up the contemporary knowledge of such signaling mechanisms, including their therapeutic targets and betokens that novel and efficacious therapies can be developed only by the keen understanding of their character in hepatocarcinogenesis. In additament, we address the role of consequential therapeutic agents and preclinical nondrug therapies known for combating hepatocarcinogenesis.

The RHO Family GTPases: Mechanisms of Regulation and Signaling.

Much progress has been made toward deciphering RHO GTPase functions, and many studies have convincingly demonstrated that altered signal transduction through RHO GTPases is a recurring theme in the progression of human malignancies. It seems that 20 canonical RHO GTPases are likely regulated by three GDIs, 85 GEFs, and 66 GAPs, and eventually interact with >70 downstream effectors. A recurring theme is the challenge in understanding the molecular determinants of the specificity of these four classes of interacting proteins that, irrespective of their functions, bind to common sites on the surface of RHO GTPases. Identified and structurally verified hotspots as functional determinants specific to RHO GTPase regulation by GDIs, GEFs, and GAPs as well as signaling through effectors are presented, and challenges and future perspectives are discussed.

Characteristics of mouse intestinal microbiota during acute liver injury and repair following 50% partial hepatectomy.

Dysbiosis of the gut microbiota has important roles in various diseases and pathological states of the host. However, the changes of the gut microbiota during partial hepatectomy (PH)-induced acute liver injury have so far remained elusive. The present study investigated the gut microbiome and its related pathways following PH-induced acute liver injury. A total of 50 male C57/BL6 mice were divided into a normal control (NC), sham-operation and liver resection (LR) group (50% PH). Samples were collected at 3 and 14 days post-operation to obtain specimens for the Sham3, Sham14, LR3 and LR14 groups (10 mice/group). Specimens of NC group (n=10) were obtained at the same time as those of Sham3 group. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were determined using an automatic chemical analyzer and the gut microbiota was assessed by 16S ribosomal RNA gene sequencing of small intestinal contents. The serum levels of ALT and AST in the LR3 group were significantly increased, while those in the LR14 group were decreased again to near-normal levels. In the LR3 group, the operational taxonomic units, species richness (Chao1) and species diversity (Shannon and Simpson indices) were decreased, although without any significant difference. Furthermore, in the LR3 group, significant Cyanobacteria enrichment and Fusobacteria depletion compared with the NC and Sham3 groups was observed, while in the LR14 group, a significant depletion of the abundance of Verrucomicrobia, Chloroflexi and Deferribacteres compared to the LR3 group was obtained. The abundance of Firmicutes was increased in the LR3 group and decreased again in the LR14 group. However, the abundance of Bacteroidetes and Actinobacteria decreased in the LR3 group and increased again in the LR14 group. The alterations of the gut microbiota at the genus level were also revealed, as significant increases in Chloroplast, Curvibacter, Pelomonas, Ruminococcaceae UCG-005 and Blautia and a sharp decrease in Akkermansia and Eubacterium coprostanoligenes were caused by acute liver injury. Furthermore, functional metagenome prediction was performed by Phylogenetic Investigation of Communities by Reconstruction of Unobserved States based on the Greengenes database, revealing alterations in signal transduction, transcription and cell motility, as well as metabolism of amino acids, lipids, glucose, cofactors and terpenoids, and xenobiotics pathways. An improved understanding of the structural and functional changes of the gut microbiota following 50% PH-induced acute liver injury and repair may provide novel strategies for the recovery of hosts undergoing hepatectomy.