Active growth signaling promotes senescence and cancer cell sensitivity to CDK7 inhibition.

Tumor growth is driven by continued cellular growth and proliferation. Cyclin-dependent kinase 7's (CDK7) role in activating mitotic CDKs and global gene expression makes it therefore an attractive target for cancer therapies. However, what makes cancer cells particularly sensitive to CDK7 inhibition (CDK7i) remains unclear. Here, we address this question. We show that CDK7i, by samuraciclib, induces a permanent cell-cycle exit, known as senescence, without promoting DNA damage signaling or cell death. A chemogenetic genome-wide CRISPR knockout screen identified that active mTOR (mammalian target of rapamycin) signaling promotes samuraciclib-induced senescence. mTOR inhibition decreases samuraciclib sensitivity, and increased mTOR-dependent growth signaling correlates with sensitivity in cancer cell lines. Reverting a growth-promoting mutation in PIK3CA to wild type decreases sensitivity to CDK7i. Our work establishes that enhanced growth alone promotes CDK7i sensitivity, providing an explanation for why some cancers are more sensitive to CDK inhibition than normally growing cells.

Transcription factor UBF depletion in mouse cells results in downregulation of both downstream and upstream elements of the rRNA transcription network.

Transcription/processing of the ribosomal RNA (rRNA) precursor, as part of ribosome biosynthesis, is intensively studied and characterized in eukaryotic cells. Here, we constructed shRNA-based mouse cell lines partially silenced for the Upstream Binding Factor UBF, the master regulator of rRNA transcription and organizer of open rDNA chromatin. Full Ubf silencing in vivo is not viable, and these new tools allow further characterization of rRNA transcription and its coordination with cellular signaling. shUBF cells display cell cycle G1 delay and reduced 47S rRNA precursor and 28S rRNA at baseline and serum-challenged conditions. Growth-related mTOR signaling is downregulated with the fractions of active phospho-S6 Kinase and pEIF4E translation initiation factor reduced, similar to phosphorylated cell cycle regulator retinoblastoma, pRB, positive regulator of UBF availability/rRNA transcription. Additionally, we find transcription-competent pUBF (Ser484) severely restricted and its interacting initiation factor RRN3 reduced and responsive to extracellular cues. Furthermore, fractional UBF occupancy on the rDNA unit is decreased in shUBF, and expression of major factors involved in different aspects of rRNA transcription is severely downregulated by UBF depletion. Finally, we observe reduced RNA Pol1 occupancy over rDNA promoter sequences and identified unexpected regulation of RNA Pol1 expression, relative to serum availability and under UBF silencing, suggesting that regulation of rRNA transcription may not be restricted to modulation of Pol1 promoter binding/elongation rate. Overall, this work reveals that UBF depletion has a critical downstream and upstream impact on the whole network orchestrating rRNA transcription in mammalian cells.

VEGFR2 survival and mitotic signaling depends on joint activation of associated C3ar1/C5ar1 and IL-6R-gp130.

Purified vascular endothelial cell (EC) growth factor receptor-2 (VEGFR2) auto-phosphorylates upon VEGF-A occupation in vitro, arguing that VEGR2 confers its mitotic and viability signaling in and of itself. Herein, we show that, in ECs, VEGFR2 function requires concurrent C3a/C5a receptor (C3ar1/C5ar1) and IL-6 receptor (IL-6R)-gp130 co-signaling. C3ar1/C5ar1 or IL-6R blockade totally abolished VEGFR2 auto-phosphorylation, downstream Src, ERK, AKT, mTOR and STAT3 activation, and EC cell cycle entry. VEGF-A augmented production of C3a/C5a/IL-6 and their receptors via a two-step p-Tyk2/p-STAT3 process. Co-immunoprecipitation analyses, confocal microscopy, ligand pulldown and bioluminescence resonance energy transfer assays all indicated that the four receptors are physically interactive. Angiogenesis in murine day 5 retinas and in adult tissues was accelerated when C3ar1/C5ar1 signaling was potentiated, but repressed when it was disabled. Thus, C3ar1/C5ar1 and IL-6R-gp130 joint activation is needed to enable physiological VEGFR2 function.

The Hydroxytyrosol Induces the Death for Apoptosis of Human Melanoma Cells.

Melanoma is the most aggressive form of skin cancer and one of the most treatment-refractory malignancies. In metastatic melanoma cell lines, we analysed the anti-proliferative and pro-apoptotic potentials of a phenolic component of olive oil, the hydroxytyrosol. In particular, through MTS assay, DeadEnd™ Colorimetric TUNEL assay, Annexin V binding and PI uptake, western blot experiment, intracellular reactive oxygen species (ROS) analysis, and the cell colony assay, we showed that the hydroxytyrosol treatment remarkably reduces the cell viability inducing the death for apoptosis of melanoma cells. Moreover, we showed that the hydroxytyrosol treatment of melanoma cells leads to a significant increase of p53 and γH2AX expression, a significant decrease of AKT expression and the inhibition of cell colony formation ability. Finally, we propose that the increased amount of intracellular reactive oxygen species (ROS) that may be related to the regulation of the pathways involved in the activation of apoptosis and in the inhibition of melanoma growth could be the strategy used by hydroxytyrosol to exert its functions in melanoma. Therefore, for its role in melanoma growth inhibition, the hydroxytyrosol treatment could deeply interfere with melanoma progression as a promising therapeutic option for the treatment of this highly invasive tumour.

The Prospective Association Between Plasma Concentrations of Cellular Growth Factors and Risk of Heart Failure Mortality in Japanese Population.

BACKGROUND: Limited evidence is available on the association of insulin-like growth factors (IGFs) and risk of heart failure in population-based samples. We investigated whether serum IGFs concentrations can predict mortality from heart failure. METHODS: We conducted a nested case-control study of 39,242 subjects aged 40-79 years who participated in the JACC study, a large Japanese prospective cohort study; participants provided serum samples and were followed up for 9 years. In heart failure cases and age-, sex-, community-, and year of blood withdrawal-matched controls, we measured serum concentrations of IGF-I, IGF-II, and IGF binding protein 3 (IGFBP3) and transforming growth factor (TGF-ß1). RESULTS: During the follow-up, there were 88 heart failure deaths (44 men and 44 women). Each increment of 1 standard deviation [SD] of IGF-II (120.0 ng/mL in women and 143.7 ng/mL in men) was associated with a 47% reduced risk of mortality from heart failure; multivariable odds ratio was 0.53 (95% confidence interval [CI], 0.30-0.94, P-trend = 0.03). The multivariable odds ratio in the highest quartile of IGFBP3 serum concentrations (≥3.29 µg/mL in women and ≥3.31 µg/mL in men) compared with the lowest (<2.11 µg/mL in women and <2.56 µg/mL in men) was 0.24 (95% CI, 0.05-1.11; P-trend = 0.12). No association was found between serum concentrations of IGF-I or TGF-ß1 and risk of heart failure. CONCLUSIONS: Higher serum concentrations of IGF-II were associated with lower mortality from heart failure, which might suggest a possible role of IGF-II in the occurrence or prognosis of heart failure.

Iron for proliferation of cell lines and hematopoietic progenitors: Nailing down the intracellular functional iron concentration.

Iron is an essential nutrient which must be provided in sufficient amounts to support growth of eukaryotic cells. All organisms devote specialized pathways to ensure proper delivery. Yet, a quantitative assessment of the intra-cellular iron concentration needed to allow the cell cycle to proceed in mammalian cells is missing. Starting from iron-depleted cell lines or primary hematopoietic progenitors prepared with clinically implemented iron chelators, replenishment via transferrin and other iron sources has been quantitatively monitored through the main endogenous markers of the cellular iron status, namely proteins involved in the uptake (transferrin receptor), the storage (ferritin), and the sensing (Iron Regulatory Proteins) of iron. When correlated with measurements of iron concentrations and indicators of growth, this minimally intrusive approach provided an unprecedented estimate of the intracellular iron concentration acting upon iron-centered regulatory pathways. The data were analyzed with the help of a previously developed theoretical treatment of cellular iron regulation. The minimal cellular iron concentration required for cell division was named functional iron concentration (FIC) to distinguish it from previous estimates of the cellular labile iron. The FIC falls in the low nanomolar range for all studied cells, including hematopoietic progenitors. These data shed new light on basic aspects of cellular iron homeostasis by demonstrating that sensing and regulation of iron occur well below the concentrations requiring storage or becoming noxious in pathological conditions. The quantitative assessment provided here is relevant for monitoring treatments of conditions in which iron provision must be controlled to avoid unwanted cellular proliferation.

Inhibition of cellular proliferation and enhancement of hydrogen peroxide production in fibrosarcoma cell line by weak radio frequency magnetic fields.

This study presents experimental data for the effects of weak radio frequency (RF) magnetic fields on hydrogen peroxide (H2O2) production and cellular growth rates of fibrosarcoma HT1080 cells in vitro. Cells were exposed either to 45 µT static magnetic fields (SMFs)-oriented vertical to the plane of growth or to SMFs combined with weak 5 and 10 MHz RF magnetic fields of 10 µTRMS intensity perpendicular to the static field. Cell numbers were reduced up to 30% on Day 2 for the cells exposed to the combination of SMF and a 10 MHz RF magnetic field compared with the SMF control cells. In addition, cells exposed to 10 MHz RF magnetic fields for 8 h increased H2O2 production by 55%. The results demonstrate an overall magnetic field-induced biological effect that shows elevated H2O2 levels with accompanying decrease in cellular growth rates.

Assessing the Effects of Curcumin and 450 nm Photodynamic Therapy on Oxidative Metabolism and Cell Cycle in Head and Neck Squamous Cell Carcinoma: An In Vitro Study.

Oral cancer is the 16th most common malignant tumor worldwide. The risk of recurrence and mortality is high, and the survival rate is low over the following five years. Recent studies have shown that curcumin causes apoptosis in tumor cells by affecting FoF1-ATP synthase (ATP synthase) activity, which, in turn, hinders cell energy production, leading to a loss of cell viability. Additionally, irradiation of curcumin within cells can intensify its detrimental effects on cancer cell viability and proliferation (photodynamic therapy). We treated the OHSU-974 cell line, a model for human head and neck squamous cell carcinoma (HNSCC), and primary human fibroblasts. The treatment involved a 1 h exposure of cells to 0.1, 1.0, and 10 µM curcumin, followed or not by irradiation or the addition of the same concentration of pre-irradiated curcumin. Both instances involved a diode laser with a wavelength of 450 nm (0.25 W, 15 J, 60 s, 1 cm2, continuous wave mode). The treatment with non-irradiated 1 and 10 µM curcumin caused ATP synthase inhibition and a consequent reduction in the oxygen consumption rate (OCR) and the ATP/AMP ratio, which was associated with a decrement in lipid peroxidation accumulation and a slight increase in glutathione reductase and catalase activity. By contrast, 60 s curcumin irradiation with 0.25 W-450 nm caused a further oxidative phosphorylation (OxPhos) metabolism impairment that induced an uncoupling between respiration and energy production, leading to increased oxidative damage, a cellular growth and viability reduction, and a cell cycle block in the G1 phase. These effects appeared to be more evident when the curcumin was irradiated after cell incubation. Since cells belonging to the HNSCC microenvironment support tumor development, curcumin's effects have been analyzed on primary human fibroblasts, and a decrease in cell energy status has been observed with both irradiated and non-irradiated curcumin and an increase in oxidative lipid damage and a slowing of cell growth were observed when the curcumin was irradiated before or after cellular administration. Thus, although curcumin displays an anti-cancer role on OHSU-974 in its native form, photoactivation seems to enhance its effects, making it effective even at low dosages.

A coarse-grained resource allocation model of carbon and nitrogen metabolism in unicellular microbes.

Coarse-grained resource allocation models (C-GRAMs) are simple mathematical models of cell physiology, where large components of the macromolecular composition are abstracted into single entities. The dynamics and steady-state behaviour of such models provides insights on optimal allocation of cellular resources and have explained experimentally observed cellular growth laws, but current models do not account for the uptake of compound sources of carbon and nitrogen. Here, we formulate a C-GRAM with nitrogen and carbon pathways converging on biomass production, with parametrizations accounting for respirofermentative and purely respiratory growth. The model describes the effects of the uptake of sugars, ammonium and/or compound nutrients such as amino acids on the translational resource allocation towards proteome sectors that maximized the growth rate. It robustly recovers cellular growth laws including the Monod law and the ribosomal growth law. Furthermore, we show how the growth-maximizing balance between carbon uptake, recycling, and excretion depends on the nutrient environment. Lastly, we find a robust linear correlation between the ribosome fraction and the abundance of amino acid equivalents in the optimal cell, which supports the view that simple regulation of translational gene expression can enable cells to achieve an approximately optimal growth state.

Chronic Kidney Disease Interplay with Comorbidities and Carbohydrate Metabolism: A Review.

Chronic kidney disease (CKD) poses a global health challenge, engendering various physiological and metabolic shifts that significantly impact health and escalate the susceptibility to severe illnesses. This comprehensive review delves into the intricate complexities of CKD, scrutinizing its influence on cellular growth homeostasis, hormonal equilibrium, wasting, malnutrition, and its interconnectedness with inflammation, oxidative stress, and cardiovascular diseases. Exploring the genetic, birth-related, and comorbidity factors associated with CKD, alongside considerations of metabolic disturbances, anemia, and malnutrition, the review elucidates how CKD orchestrates cellular growth control. A pivotal focus lies on the nexus between CKD and insulin resistance, where debates persist regarding its chronological relationship with impaired kidney function. The prevalence of insulin abnormalities in CKD is emphasized, contributing to glucose intolerance and raising questions about its role as a precursor or consequence. Moreover, the review sheds light on disruptions in the growth hormone and insulin-like growth factor axis in CKD, underscoring the heightened vulnerability to illness and mortality in cases of severe growth retardation. Wasting, a prevalent concern affecting up to 75% of end-stage renal disease (ESRD) patients, is analyzed, elucidating the manifestations of cachexia and its impact on appetite, energy expenditure, and protein reserves. Taste disturbances in CKD, affecting sour, umami, and salty tastes, are explored for their implications on food palatability and nutritional status. Independent of age and gender, these taste alterations have the potential to sway dietary choices, further complicating the management of CKD. The intricate interplay between CKD, inflammation, oxidative stress, and cardiovascular diseases is unraveled, emphasizing the profound repercussions on overall health. Additionally, the review extends its analysis to CKD's broader impact on cognitive function, emotional well-being, taste perception, and endothelial dysfunction. Concluding with an emphasis on dietary interventions as crucial components in CKD management, this comprehensive review navigates the multifaceted dimensions of CKD, providing a nuanced understanding essential for developing targeted therapeutic strategies.

Upregulation of ribosome biogenesis via canonical E-boxes is required for Myc-driven proliferation.

The transcription factor Myc drives cell growth across animal phyla and is activated in most forms of human cancer. However, it is unclear which Myc target genes need to be regulated to induce growth and whether multiple targets act additively or if induction of each target is individually necessary. Here, we identified Myc target genes whose regulation is conserved between humans and flies and deleted Myc-binding sites (E-boxes) in the promoters of fourteen of these genes in Drosophila. E-box mutants of essential genes were homozygous viable, indicating that the E-boxes are not required for basal expression. Eight E-box mutations led to Myc-like phenotypes; the strongest mutant, ppanEbox-/-, also made the flies resistant to Myc-induced cell growth without affecting Myc-induced apoptosis. The ppanEbox-/- flies are healthy and display only a minor developmental delay, suggesting that it may be possible to treat or prevent tumorigenesis by targeting individual downstream targets of Myc.

Comparison of Different Chemical and Mechanical Modalities for Implant Surface Decontamination: Activity against Biofilm and Influence on Cellular Regrowth-An In Vitro Study.

Objectives: The aim of this in vitro study was to compare the efficacy of chemical and mechanical methods for decontamination of titanium dental implant surfaces previously infected with polymicrobial biofilms in a model simulating a peri-implant defect. Furthermore, the effect of each decontamination protocol on MG-63 osteoblast-like cells morphology and adhesion to the treated implants was assessed. Background: Peri-implantitis is a growing issue in dentistry, and evidence about implant surface decontamination procedures is lacking and inconclusive. Methods: A total of 40 previously biofilm-contaminated implants were placed into a custom-made model simulating a peri-implant defect and randomly assigned to five treatment groups: (C) control (no treatment); (AW) air abrasion without any powder; (ESC) air abrasion with powder of erythritol, amorphous silica, and 0.3% chlorhexidine; (HBX) decontamination with a sulfonic/sulfuric acid solution in gel; and (HBX + ESC) a combination of HBX and ESC. Microbiological analysis was performed on five implants per treatment group, and the residual viable bacterial load measured in log 10 CFU/mL was counted for each bacterial strain and for the total number of colonies. The remaining three implants per group and three noncontaminated (NC) implants were used to assess surface biocompatibility using a scanning electron microscope and a backscattered electron microscope after seeding with MG-63 cells. Results: A significant decontaminant effect was achieved using HBX or HBX + ESC, while no differences were observed among other groups. The percentage of implant surface covered by adherent MG-63 cells was influenced by the treatment method. Progressive increases in covered surfaces were observed in groups C, AW, ESC, HBX, HBX + ESC, and NC. Conclusions: A combination of mechanical and chemical decontamination may provide more predictable results than mechanical cleaning alone.

Effects of Dexamethasone on DNA Synthesis in Lens Epithelial Cells Are Dependent on Cell Type and Growth Factor.

PURPOSE: To determine the factors that influence the ability of dexamethasone (dex) to inhibit or stimulate the growth of lens epithelial cells. METHOD: Different growth factors with or without dex (10-6 M) were added to quiescent cultures of two clones of Nakano mouse lens epithelial cells (NK11) in serum-free medium. DNA synthesis was then measured after 8-12 hours by the incorporation of tritiated thymidine. RESULTS: Dex was found to both stimulate and inhibit mitogen-induced 3H-thymidine incorporation into the DNA of cultured mouse lens epithelial cells. Enhancement or repression by dex was found to depend on the growth factor used to stimulate the quiescent cell. EGF and insulin were consistently inhibited with dex. Basic fibroblast growth factor (bFGF) and retinoblastoma-derived growth factor (RbDGF) were both enhanced and inhibited by dex, depending on the growth factor concentration and the cell clone used for the experiment. Additionally, RbDGF protects against the dex inhibition of insulin stimulation, but not the inhibition of EGF stimulation. Progesterone, an inhibitor of the activation of the glucocorticoid receptor, blocks the dex inhibitory effect on the EGF and insulin stimulation of DNA synthesis. The ability of progesterone to affect the dex inhibition is consistent with the dex receptor modulating DNA synthesis. The dex effect on DNA synthesis, either stimulatory or inhibitory, was still seen if dex was added as late as 10 hours after the growth factor. CONCLUSIONS: The study demonstrated that dex reduces the overall growth and activity of lens epithelial cells in vitro. This result provides insight into the risk of developing posterior subcapsular cataracts (PSC) in patients on oral glucocorticoid therapy. Understanding the basic mechanisms by which steroids mediate lens cell growth may provide the ability to more accurately predict who will develop PSC. The present studies show the difference in the effect of dex from lens cell to lens cell, but, more importantly, suggest a pattern of dependent variables that might prove useful in such predictions.

Fundamental limits on the rate of bacterial growth and their influence on proteomic composition.

Despite abundant measurements of bacterial growth rate, cell size, and protein content, we lack a rigorous understanding of what sets the scale of these quantities and when protein abundances should (or should not) depend on growth rate. Here, we estimate the basic requirements and physical constraints on steady-state growth by considering key processes in cellular physiology across a collection of Escherichia coli proteomic data covering ≈4,000 proteins and 36 growth rates. Our analysis suggests that cells are predominantly tuned for the task of cell doubling across a continuum of growth rates; specific processes do not limit growth rate or dictate cell size. We present a model of proteomic regulation as a function of nutrient supply that reconciles observed interdependences between protein synthesis, cell size, and growth rate and propose that a theoretical inability to parallelize ribosomal synthesis places a firm limit on the achievable growth rate. A record of this paper's transparent peer review process is included in the supplemental information.

Quantifying Cyanothece growth under DIC limitation.

The photoautotrophic, unicellular N2-fixer, Cyanothece, is a model organism that has been widely used to study photosynthesis regulation, the structure of photosystems, and the temporal segregation of carbon (C) and nitrogen (N) fixation in light and dark phases of the diel cycle. Here, we present a simple quantitative model and experimental data that together, suggest external dissolved inorganic carbon (DIC) concentration as a major limiting factor for Cyanothece growth, due to its high C-storage requirement. Using experimental data from a parallel laboratory study as a basis, we show that after the onset of the light period, DIC was rapidly consumed by photosynthesis, leading to a sharp drop in the rate of photosynthesis and C accumulation. In N2-fixing cultures, high rates of photosynthesis in the morning enabled rapid conversion of DIC to intracellular C storage, hastening DIC consumption to levels that limited further uptake. The N2-fixing condition allows only a small fraction of fixed C for cellular growth since a large fraction was reserved in storage to fuel night-time N2 fixation. Our model provides a framework for resolving DIC limitation in aquatic ecosystem simulations, where DIC as a growth-limiting factor has rarely been considered, and importantly emphasizes the effect of intracellular C allocation on growth rate that varies depending on the growth environment.

Logistic Models for Simulating the Growth of Plants by Defining the Maximum Plant Size as the Limit of Information Flow.

Today, the Logistic equations are widely applied to simulate the population growth across a range of fields, chiefly, demography and ecology. Based on an assumption that growth-regulating factors within the Logistic model, namely, the rate of increase (r) and carrying capacity (K), can be considered as the functions reflecting the combination of the organism- and environment-specific parameters, here, we discussed the possible application of modified synthetic Logistic equations to the simulation of the changes in (1) population (density per volume) of photosynthetically growing free-living algae and (2) size (mass per individual) of higher plants, by newly composing r value as a function reflecting the photosynthetic activities. Since higher plants are multi-cellular organisms, a novel concept for the carrying capacity K must also be introduced. We brought the a priori assumption that information sharing amongst cells strongly influences the physiology of multi-cellular structures eventually defining the maximum size of plants, into view. A simplest form of 'synthetic organism' conformed to test this assumption is a linear chain of cells, and the first physiological phenomenon, modeled in this way, is growth. This combination of information flow along a chain, with exponential growth, produces a simple allotropic relationship. This relationship is compared with results for plants and is found to have excellent predictive power. This theory shows that fast-growing organisms, or multicellular structures, remain small, because of their inability to share information sufficiently quickly and, also, predicts determinate growth. The success of this simple model suggests, firstly, that the inclusion of information flows in theoretical physiology models, which have been, to date, dominated by energetic or metabolic assumptions, will be improved by incorporating information flows. Secondly, the application of more complex information theories, such as those of Shannon, to biological systems will offer deep insights into the mechanisms and control of intercellular communication.

Nutrient supplementation strategy improves cell concentration and longevity, monoclonal antibody production and lactate metabolism of Chinese hamster ovary cells.

A careful selection of culture mediums and feeds has become necessary to maximize yields of recombinant proteins during bioprocesses of mammalian cells. Supplements contain a variety of concentrate nutrients, and their beneficial effects vary according to recombinant cell lines. In this study, the effects of PowerFeed A on growth kinetics, productivity and cellular metabolism were evaluated for two Chinese hamster ovary cell lines producing a monoclonal antibody in a batch culture. Supplemented cultures increased integral viable cell density of CRL-12444 and CRL-12445 cells by 2.4 and 1.6 times through extension of culture time at which viability was above 90% in 72 and 36 h, respectively, and increment of maximal cell concentration in 3.25 × 106 cells/ml (69%) for CRL-12445 cells. Product titer augmented 1.9 and 2.5 times for CRL-12444 and CRL-12445 cells, respectively, without changes in growth rate and specific productivity. Feed supplementation also stimulated full consumption of glucose and free glutamine and reduced 10 times lactate accumulation, while ammonium, sodium and potassium remained at similar concentrations at the end of the culture. About 44% of calcium, mainly provided by feed, was consumed by both cell lines. Maximization of cellular growth, viability and protein titer through feeding encourages extending its use to other cell lines and exploring novel combinations with other basal mediums or feeds. A thorough investigation of its impact on protein quality and the molecular mechanisms behind these effects will allow designing effective feeds and strategies to rationally optimize protein production in the biomanufacturing industry.

Dibutyltin(IV) and Tributyltin(IV) Derivatives of meso-Tetra(4-sulfonatophenyl)porphine Inhibit the Growth and the Migration of Human Melanoma Cells.

Melanoma is the most aggressive and deadly form of skin cancer, which is largely due to its propensity to metastasize. Therefore, with the aim to inhibit the growth and the metastatic dissemination of melanoma cells and to provide a novel treatment option, we studied the effects of the melanoma treatment with two organotin(IV) complexes of the meso-tetra(4-sulfonato-phenyl)porphine, namely (Bu2Sn)2TPPS and (Bu3Sn)4TPPS. In particular, we showed that nanomolar concentrations of (Bu2Sn)2TPPS and (Bu3Sn)4TPPS are sufficient to inhibit melanoma cell growth, to increase the expression of the full-length poly (ADP-ribose) polymerase (PARP-1), to induce the cell cycle arrest respectively at G2/M and G0/G1 through the inhibition of the Cyclin D1 expression and to inhibit cell colony formation. Nanomolar concentrations of (Bu2Sn)2TPPS and (Bu3Sn)4TPPS are also sufficient to inhibit the melanoma cell migration and the expression of some adhesion receptors. Moreover, we report that (Bu2Sn)2TPPS and (Bu3Sn)4TPPS act downstream of BRAF, mainly bypassing its functions, but targeting the STAT3 signalling protein. Finally, these results suggest that (Bu2Sn)2TPPS and (Bu3Sn)4TPPS may be effective therapeutic strategies for their role in the inhibition of melanoma growth and migration.

UBE2C, Directly Targeted by miR-548e-5p, Increases the Cellular Growth and Invasive Abilities of Cancer Cells Interacting with the EMT Marker Protein Zinc Finger E-box Binding Homeobox 1/2 in NSCLC.

Background: Recent evidence indicates that UBE2C participates in carcinogenesis by regulating the cell cycle, apoptosis, metastasis, and transcriptional processes. Additionally, miR-548e-5p dysregulation plays a vital role in tumor progression. However, the molecular mechanism via which UBE2C is directly targeted by miR-548-5p, resulting in increase in cellular growth and invasiveness of cancer cells, and its interactions with the epithelial-mesenchymal transition (EMT) marker protein ZEB1/2 in non-small cell lung cancer (NSCLC) is not understood. Methods: Expression of UBE2C and miR-548e-5p was analyzed using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The protein level of UBE2C and ZEB1/2 was analyzed using western blotting and immunofluorescence staining. Cellular proliferation was detected using the cell counting kit 8 (CCK8) and 3-(4,5-dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium bromide (MTT) assays. Cell migration, invasion, and growth were analyzed using the wound healing and transwell assay. Promoter activity and transcription was analyzed using the luciferase reporter assay. Chromatin immunoprecipitation was used to detect binding of UBE2C to 5'UTR-ZEB1/2. Results: We observed that 4,5-ubiquitin-conjugating enzyme E2C (UBE2C) expression was higher in NSCLC tissue than in the adjacent normal tissue and was associated with increased cell proliferation and invasion. UBE2C enhanced NSCLC progression and metastasis by affecting the cell cycle and inhibiting apoptosis. We also observed that miR-548e-5p was significantly downregulated in lung cancer tissue specimens, which decreased the expression of its direct substrate, UBE2C. Moreover, miR-548e-5p overexpression and UBE2C under-expression significantly suppressed lung cancer cell proliferation, migration, and invasion. Luciferase reporter and chromatin immunoprecipitation assays indicated that miR-548e-5p directly binds to the 3'-UTR of UBE2C and decreases UBE2C mRNA expression. Furthermore, UBE2C knockdown downregulated the mesenchymal marker vimentin and upregulated the epithelial marker E-cadherin. Bioinformatics assays, coupled with western blotting and luciferase assays, revealed that UBE2C directly binds to the 5'-untranslated region (UTR) of the transcript of the E-cadherin repressor ZEB1/2 and promotes EMT in lung cancer cells. Conclusion: miR-548e-5p directly binds to the 3'-UTR of UBE2C and decreases UBE2C mRNA expression. UBE2C is an oncogene that promotes EMT in lung cancer cells by directly targeting the 5'-UTR of the transcript encoding the E-cadherin repressor ZEB1/2. miR-548e-5p, UBE2C, and ZEB1/2 constitute the miR-548e-5p-UBE2C-ZEB1/2 signal axis, which enhances cancer cell invasiveness by directly interacting with e EMT marker proteins. We believe that the miR-548e-5p-UBE2C-ZEB1/2 signal axis may be a suitable diagnostic marker and a potential target for lung cancer therapy.