D1 Levels Research Articles

Grain yield and interspecific competition in an oat-common vetch intercropping system at varying sowing density.

Oat (Avena nuda L.) and common vetch (Vicia sativa L.) intercropping in the northern regions of China has resulted in substantial production capabilities. However, there is currently a dearth of comprehensive research on whether this intercropping system can enhance productivity through increased sowing densities and underlying interspecies interaction mechanisms. A two-year field experiment was conducted in 2022 and 2023 to investigate the yield, biological efficiency, economic efficiency, and competition indicators of oats and common vetch in a high-density intercropping system. Two cropping patterns (monocropping and intercropping) and five sowing densities (D1: 4.5×106 plants ha-1; D2:5.4×106 plants ha-1; D3:6.3×106 plants ha-1; D4: 7.2×106 plants ha-1; and D5: 8.1×106 plants ha-1) were arranged in a randomized block design. At the same sowing density, the intercropped oats exhibited greater grain yield than the monocultures. Increasing the oat sowing density significantly enhanced oat yield, with the D3 level in intercropping showing the highest yield increase, ranging from 30.98% to 31.85%, compared with the monoculture. The common vetch intercropping grain yield was maximized in the D2 treatment. The land equivalent ratio was maximized at the D2 level in both years and was significantly higher than D1, with the land equivalent coefficient, system productivity index, and percentage yield difference suggesting that increasing oat sowing densities improved the productivity of the intercropping system, with the best performance observed at the D2 level. For both years, the proportionate actual yield loss of oat was the highest at the D3 level; significantly surpassing D1, proportionate actual yield loss of common vetch and actual yield loss were the highest at level D2, both significantly surpassing D1. These indicates that appropriate densification contributes to the realization of the advantages of intercropping. With an increased oat sowing density, the economic benefits of the intercropping system were maximized at the D2 and D3 levels. Regarding intercropping competition, oat was the dominant crop under different sowing densities (Aggressivity for oat (AO)>0, relative crowding coefficient for oat (KO)>1, competition ratio for oat (CRO)>1), whereas common vetch was the inferior crop. Compared with the D1 level, the D2 level harmonized the aggressivity, competitive ratio, and relative crowding coefficients of oat and common vetch, significantly increasing crowding coefficient for common vetch (KV) and competition ratio for common vetch by 19.76% to 21.94% and 4.80% to 7.51%, respectively, while reducing KO and CRO. This result suggests that in the intercropping of common vetch and oat in alpine regions, rational densification can harmonize interspecific competition and thus improve the biological efficiency and economic benefits of intercropping systems.

Reflecting on Reflective Journals of Indonesian EFL Pre-Service Teachers to Examine Their Quality of Reflective Practice

Reflective practice is essential for teachers’ professional development. It leads teacher to shape and reshape their knowledge, and so forth a wide range of benefits can be achieved. However, to gain such benefits, teachers must have good quality of reflective practice. This qualitative case study aimed at investigating quality of reflective practice of four female EFL pre-service teachers in Indonesia. Framed with qualitative case study, this study employed online reflective journals as the main instrument to collect the data. The data were analysed deductively using content analysis. The results revealed that participants’ reflection tended to remain in the level of D1 and D2 which meant that they had tendency to merely retell and describe their teaching practice with limited evaluation. Only few of them were captured to reflect on the level of R1 and R2. In sum, although Indonesian EFL pre-service teachers had tendency to reflect in low level, it was interesting to find the fact that they sometimes reflected more critically in the level of R1 and R2. The result could be used as indicator that Indonesian EFL pre-service teachers had ability to reflect in a good way. Thus, following actions by related parties should be established in order to develop their abilities.

The State of the Dopaminergic and Glutamatergic Systems in the Valproic Acid Mouse Model of Autism Spectrum Disorder.

Autism Spectrum Disorder (ASD) is a progressive neurodevelopmental disorder mainly characterized by deficits in social communication and stereotyped behaviors and interests. Here, we aimed to investigate the state of several key players in the dopamine and glutamate neurotransmission systems in the valproic acid (VPA) animal model that was administered to E12.5 pregnant females as a single dose (450 mg/kg). We report no alterations in the number of mesencephalic dopamine neurons or in protein levels of tyrosine hydroxylase in either the striatum or the nucleus accumbens. In females prenatally exposed to VPA, levels of dopamine were slightly decreased while the ratio of DOPAC/dopamine was increased in the dorsal striatum, suggesting increased turn-over of dopamine tone. In turn, levels of D1 and D2 dopamine receptor mRNAs were increased in the nucleus accumbens of VPA mice suggesting upregulation of the corresponding receptors. We also report decreased protein levels of striatal parvalbumin and increased levels of p-mTOR in the cerebellum and the motor cortex of VPA mice. mRNA levels of mGluR1, mGluR4, and mGluR5 and the glutamate receptor subunits NR1, NR2A, and NR2B were not altered by VPA, nor were protein levels of NR1, NR2A, and NR2B and those of BDNF and TrkB. These findings are of interest as clinical trials aiming at the dopamine and glutamate systems are being considered.

Fucoxanthinol Promotes Apoptosis in MCF-7 and MDA-MB-231 Cells by Attenuating Laminins–Integrins Axis

Fucoxanthinol (FxOH), the main metabolite of the marine carotenoid fucoxanthin, exerts anti-cancer effects. However, fragmentary information is available on the growth-inhibiting effects of FxOH on breast cancer (BC). We investigated the growth-inhibiting effects of FxOH on human BC cells (MCF-7 and MDA-MB-231 cells), and the underlying mechanisms, differently from previous studies, by using comprehensive transcriptome analysis. The molecular mechanisms of FxOH were evaluated using flow cytometry, microarray, Western blotting, and gene knockdown analyses. FxOH (20 μM) significantly induced apoptosis in MCF-7 and MDA-MB-231 cells. Transcriptome analysis revealed that FxOH modulated the following 12 signaling pathways: extracellular matrix (ECM), adhesion, cell cycle, chemokine and cytokine, PI3K/AKT, STAT, TGF-β, MAPK, NF-κB, RAS/Rho, DNA repair, and apoptosis signals. FxOH downregulated the levels of laminin β1, integrin α5, integrin β1, integrin β4, cyclin D1, Rho A, phosphorylated (p)paxillin (Tyr31), pSTAT3(Ser727), and pSmad2(Ser465/467), which play critical roles in the 12 signaling pathways mentioned above. Additionally, FxOH upregulated the levels of pERK1/2(Thr202/Tyr204) and active form of caspase-3. Integrin β1 or β4 knockdown significantly inhibited the growth of MCF7 and MDA-MB-231 cells. These results suggest that FxOH induces apoptosis in human BC cells through some core signals, especially the ECM–integrins axis, and the downstream of cell cycle, STAT, TGF-β, RAS/Rho, MAPK, and/or DNA repair signals.

CHREBP suppresses gastric cancer progression via the cyclin D1-Rb-E2F1 pathway

Accumulating evidence has demonstrated that carbohydrate response element binding protein (CHREBP) has a crucial function in tumor pathology. In this study, we found CHREBP downregulation in gastric cancer (GC) tissues, and CHREBP was determined to be an independent diagnostic marker of GC. The downregulation of CHREBP promoted cell proliferation and inhibited apoptosis. Moreover, the level of cyclin D1 was significantly correlated with CHREBP expression in GC and paracancerous normal samples. In addition, CHREBP transcriptionally inhibited cyclin D1 expression in GC cells. Tumor suppressor activity of CHREBP could be affected by the upregulation of cyclin D1. In summary, CHREBP was found to be an independent diagnostic marker of GC and to influence GC growth and apoptosis via targeting the cyclin D1-Rb-E2F1 pathway.

Abstract 4067: Combination venetoclax and selinexor effective in relapsed refractory multiple myeloma with translocation t(11;14)

Abstract Translocation t(11;14) positive multiple myeloma (MM) is sensitive to venetoclax, yet the drug lacks FDA approval in MM. Selinexor is an inhibitor of nuclear exporter XPO1 and is approved in relapsed refractory MM. We recently analyzed RNA-seq from 859 MM patient samples (MMRF CoMMpass study) and compared them to healthy bone marrow samples (GSE114922). After correcting for batch effects, differential gene expression analysis found apoptosis and cell cycle regulation amongst the top upregulated gene pathways in MM. We therefore hypothesized that MM would be sensitive to the combination of the pro-apoptotic drug, venetoclax, and selinexor, which causes cell cycle arrest. Using a panel of 8 MM cell lines (U266-B1, KMS12BM, SKMM2, RPMI-8226, LP1, OPM2, KMS12PE, and NCIH929), we performed cell viability assays in increasing concentrations of selinexor, venetoclax, and a combination of the two drugs at 72 hours. Overall, the combination showed synergy in MM cell lines but strikingly, cell lines that possessed t(11;14) were more sensitive to the drug combination and showed enhanced synergy compared to the non-t(11;14). Average Bliss model synergy score were 0.5 in non-t(11;14) and 10.2 in t(11;14) MM cells (>10 indicates synergistic effects and <-10 indicates antagonistic drug effects). To better understand molecular mechanisms underlying the observed synergistic effect in t(11;14), we performed western blot analysis in these cell lines treated with selinexor, venetoclax, the combination, or DMSO control for 24 hours. We measured protein levels of Cyclin D1, which is overexpressed in t(11;14) and a cargo of XPO1. We confirmed Cyclin D1 overexpression in t(11;14) cells lines but not in non-t(11;14) cells. Cyclin D1 levels decreased with selinexor, and the reduction was enhanced by adding venetoclax. Additionally, we measured levels of XPO1, p53, MCL-1, and p65, which we have previously shown to be altered by selinexor treatment. While venetoclax monotherapy caused increased MCL-1 expression, this was abrogated by combining with selinexor and the effects were statistically more significant in t(11;14) cell lines. Concurrently, two patients with relapsed, refractory t(11;14) MM and disease progression after multiple lines of therapy were treated with venetoclax; however, after initial response, they developed resistance and progressive disease. Based off our preclinical results, selinexor was added to venetoclax and recaptured responses (VGPR and MR, respectively) suggesting a beneficial effect of the combination over single agent venetoclax. To our knowledge, this is the first report of patients with MM treated with the combination of selinexor and venetoclax. Further clinical evaluation of this combination in t(11;14) MM is planned and samples will be collected to better understand the mechanistic correlation between XPO1, BCL2/MCL-1, and Cyclin D1 levels and response. Citation Format: Sana Chaudhry, Nina Nguyen, Rimpi Khurana, Tulasigeri M. Totiger, Skye Montoya, Jumana Afaghani, Terrence Bradley, Francesco Maura, Dickran Kazandjian, Jennifer Chapman, Stephan Schürer, Ola Landgren, James Hoffman, Justin Taylor. Combination venetoclax and selinexor effective in relapsed refractory multiple myeloma with translocation t(11;14) [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 4067.

Abstract 6332: Pegylated arginase isolated from beef liver tissue inhibited cell proliferation, induced apoptosis & inhibits induced mammary tumors in human breast cancer in vitro & in vivo

Abstract Arginase, an enzyme of the urea cycle, converts L-arginine to L-ornithine, which is the precursor of polyamines that are essential components of cell proliferation. However, Arginine deprivation and Arginase clinical implications in human breast and liver cancers have not been fully elucidated. The objective of the current study was to investigate the potential role of the Arginine degrading enzyme, Arginase, on three cell lines; Luminal A breast cancer (T-47D), TNBC (MDA-MB-231), and HCC cell line, HepG2, respectively. Herein, Arginase was extracted and purified from the beef liver and its specific activity was 0.2 IU/mg protein yielded 49% compared to the crude beef liver Arginase. The purified Arginase is covalently bound via a succinamide propionic acid (SPA) linker to polyethylene glycol (PEG) of molecular weight 5,000 to enhance the half-life time compared to the native enzyme with a maximum specific activity between 37~55℃ and pH 7~9. The potential influence of extracted Peg-Arginase was examined on cell proliferation, clonogenic assays, morphological alteration, and oncogenic signaling pathways of STAT3, β-catenin, and miR-23a. Our data showed that Peg-Arginase treatment displayed a significant inhibition in cell proliferation and viability in a dose-dependent manner in both breast cancer cell lines, but not in HepG2 cells after 48h. Furthermore, mechanistic studies showed that Peg-Arginase induced apoptosis that was associated with cell morphological changes, histone release from fragmented DNA, caspase-3 activation in a dose-dependent manner after 48h, and cell cycle arrest at S and/or G2/M phases. Moreover, Peg-Arginase down-regulated miR-23a, STAT3, β-catenin, cyclin D1, and mutant p53 levels as well as increased p21and Bax, pro-apoptotic signal, in both breast cancer cells as determined by flow cytometry and western blot analysis. In vivo, the effects of native- and Peg-Arginase enzymes were examined on MNU-induced mammary tumors in Albino-Wister rats. Our data showed that 400 IU of native- or peg- Arginase twice per week for two weeks significantly inhibited the growth of MNU-induced mammary tumors by 58.8±4.1 % for peg-Arginase compared to 19.7±3.5 % for native-Arginase. Furthermore, the results also revealed a significant reduction in activities of ALT, AST, and ALP in the Peg-Arginase group compared to MNU untreated or native-Arginase groups, suggesting that Peg-Arginase may eliminate the hepatotoxic effect of the MNU. All in all, our results suggested that the arginine-depleting enzyme, Peg-Arginase, exerted an anticancer effect against both T-47D and MDA-MB-231 breast cancer cell lines through cell proliferation inhibition and apoptosis induction. Citation Format: Asmaa Ali, Omnia Ali, Doaa Othman, Ahmed Sultan. Pegylated arginase isolated from beef liver tissue inhibited cell proliferation, induced apoptosis & inhibits induced mammary tumors in human breast cancer in vitro & in vivo [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 6332.

Abstract 2221: Molecular analysis finds excision margin width predictive of recurrence in acral melanoma

Abstract Surgical excision with histologically clear margins results in cure of most melanoma patients. Involvement of the margins is associated with tumour recurrence and adverse outcomes. Determining whether or not the histological margins are involved by melanoma, particularly in situ melanoma, can be very difficult particularly in acral locations where insitu melanoma can be extremely subtle. This study sought to determine the proximity of acral melanoma in situ to the surgical excision margin using multiple methods including routine histopathological assessment, FISH (CCND1 amplification), and multiplex immunofluorescence (mIF: SOX10, p16, Cyclin D1 and PRAME) in primary acral melanomas with initially histopathologically reported clear excision margins of >0.3mm (n=85). The clear excision margins determined by each modality were compared between those melanomas which recurred (n=47) and those which remained recurrence-free at last follow-up (median = 46 months, range = 1-260 months) (n=35). Histopathologically confirmed excision margin width was smaller in the tumours of patients who recurred (mean = 7.083mm vs 9.964mm, p = 0.0811). Intratumoural CCND1 amplification was increased in recurrence-free patients in comparison to recurrence patients (16.2% vs 7.7%). FISH and mIF confirmed the correlation of intratumoural CCND1 amplification with Cyclin D1 expression SOX10+ melanoma cells (p=0.0347). No correlations between recurrence status and Cyclin D1, PRAME or p16 expression levels in the tumour or epidermis were identified. In histopathologically normal epidermal tissue as assessed on H&E sections,Cyclin D1+, p16- and PRAME+ cells were classified as malignant cells when co-expressed with SOX10. Upon measuring the distance from the tumour margin to the nearest malignant cell, we found that malignant cells were closer to the tumour in recurrence patients in comparison to recurrence-free patients (mean = 7.324mm vs 9.464mm, p = 0.2735). These distances closely correlated with the excision margin width as determined via histopathological assessment (R2 = 0.7424, p<0.0001). In conclusion, histological assessment, FISH and mIF determined excision margins are highly correlative in acral melanomas. Narrower excision margins were associated with local recurrence of melanoma. Citation Format: Grace Heloise Attrill, Teresa A. Bailey, Trina Lum, Jordan W. Conway, Georgina V. Long, James S. Wilmott, Richard A. Scolyer. Molecular analysis finds excision margin width predictive of recurrence in acral melanoma [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 2221.

Total Saponins of Radix Clematis Regulate Fibroblast-Like Synoviocyte Proliferation in Rheumatoid Arthritis via the LncRNA OIP5-AS1/MiR-410-3p/Wnt7b Signaling Pathway

Background Rheumatoid arthritis (RA) is the most common autoimmune disease and affects multiple joints. Previous studies have shown that total saponins of Radix clematidis (TSC) have a clear therapeutic effect on RA, but the specific mechanism has not yet been clarified. Literature screening and previous research suggest that the lncRNA OIP5-AS1/miR-410-3p/Wnt7b signaling pathway exerts a regulatory effect on the pathogenesis of RA. In this study, we examined whether the TSC treatment of RA affects the lncRNA OIP5-AS1/miR-410-3p/Wnt7b pathway. Materials and Methods Freund's complete adjuvant was used to create an adjuvant arthritis (AA) rat model with rat synovial cells being harvested and cultured. The experiment comprises a normal group, model group, TSC optimal-dose group, TSC optimal-dose group + lncRNA OIP5-AS1siRNA group, lncRNA OIP5-AS1 siRNA group, and lncRNA OIP5-AS1 siRNA + NC group. MMT was used to screen the optimal concentration of TSC. The level of lncRNA OIP5-AS1, miR-410-3p, Wnt7b, β-catenin, c-Myc, cyclin D1, GSK-3β, and SFRP4 mRNA were detected by real-time-qPCR, the expression of Wnt7b, β-catenin, c-Myc, cyclin D1, GSK-3β, and p-GSK-3β (Ser9) protein were detected by immunofluorescence and Western blot. Results We found that TSC inhibits the proliferation of RA FLS, TSC significantly reduced lncRNA OIP5-AS1, Wnt7b, β-catenin, c-Myc, cyclin D1, and p-GSK-3β/GSK-3β mRNA/protein expression, whereas the miR-410-3p and SFRP4 mRNA/protein expression levels were significantly upregulated. Our data suggest that TSC can inhibit the excessive proliferation of FLS to treat RA, the mechanism of which may be closely related to regulation of the lncRNA OIP5-AS1/miR-410-3p /Wnt7b signaling axis and the Wnt signaling pathway.

KIF14 affects cell cycle arrest and cell viability in cervical cancer by regulating the p27Kip1 pathway

BackgroundCervical cancer is a kind of malignant gynecological tumor. The first choice for treating cervical cancer is still a combination of surgery and chemoradiotherapy, but the 5-year survival rate remains poor. Therefore, researchers are trying to find new ways to diagnose and treat cervical cancer early.MethodsThe expression level of KIF14 in cells and tissues was determined via qRT–PCR. The ability of the cells to proliferate, migrate, and invade was examined using CCK-8 assay kits, colony formation assays, and Transwell chambers. The expression levels of Cyclin D1, Cyclin B1, p21, and p27 were also detected using western blot assays.ResultsThe results suggested that p27 is a key regulatory factor in the KIF14-mediated regulation of the cell cycle. In addition, KIF14 knockdown promotes malignancy in cervical cancer cells by inhibiting p27 degradation, resulting in cell cycle arrest.ConclusionsKIF14 is an oncogene in cervical cancer, and knocking down KIF14 causes cell cycle arrest by inhibiting p27 degradation, thus affecting cell viability, proliferation, and migration. These results provide a potential therapeutic target for cervical cancer.

Effects of Icodextrin Solution (Adept®) on Ovarian Cancer Cell Proliferation in an In Vitro Model.

Background and objective: Anti-adhesion barriers are currently used during ovarian cancer surgery to decrease adhesion-related morbidity. Adept® (4% icodextrin) solution, a liquid anti-adhesion material, has been widely used during gynecologic surgeries, though the risk of this barrier for oncologic surgery is controversial. The aim of this study was to determine the effect of Adept® solution on the proliferation of ovarian cancer cells. Materials and methods: We assessed the dose- and time-dependent effects of icodextrin on the growth and proliferation of OVCAR-3 and A2780 human ovarian tumor cell lines in vitro. Cell growth was determined by cell number counting. Expressions of cell cycle-regulation proteins (cyclin D1 and cyclin B1) were determined using Western blot analysis. Results: Adept® did not significantly increase ovarian cancer cell growth when tested at various concentrations (0, 1, 5, 10, 15, and 20%, equal to 0, 0.04, 0.2, 0.4, 0.6 and 0.8% icodextrin) and different time points (1–3 days) compared to control cells. Moreover, the protein levels of cyclin D1 and B1 were not overexpression-elevated in icodextrin-treated ovarian cancer cells, either with an increasing concentration or with an increasing treated time. These results demonstrated that Adept® does not activate the growth or proliferation of ovarian cancer cells in either a dose- or time-dependent manner. Conclusions: This study supports the use of Adept® solution as a safe anti-adhesion barrier for ovarian cancer surgery, though further in vivo studies are necessary.

Abstract P2-04-01: Overexpressed cyclin D1 and CDK4 proteins are responsible for the resistance to CDK4/6 inhibitor in breast cancer that can be reversed by PI3K/mTOR inhibitors

Abstract CDK4/6 inhibitors are now the standard treatment in advanced HR+/HER2- breast cancer patients. Nevertheless, the resistance to CDK4/6 inhibitors is inevitable, limiting its use and efficacy. The mechanisms and the strategies to overcome resistance are of great interest. Here, we show that the palbociclib-resistant breast cancer cells express significantly higher levels of Cyclin D1 and CDK4 proteins because of upregulated protein synthesis. Silencing Cyclin D1 or CDK4 directly led to cell cycle arrest while silencing Cyclin E1 or CDK2 restored the sensitivity to palbociclib in palbociclib-resistant cells. Furthermore, PI3K/mTOR pathway was hyper-activated in palbociclib-resistant cells, leading to more phosphorylated 4E-BP1 and higher levels of Cyclin D1 and CDK4 translation. Targeting PI3K/mTOR pathway with a specific PI3Kα inhibitor (BYL719) or an mTOR inhibitor (everolimus) reduced the protein levels of Cyclin D1 and CDK4, and restored the sensitivity to palbociclib in resistant cells both in vitro and in vivo. The tumors from breast cancer patients who progressed after palbociclib treatment had significantly higher protein expression of Cyclin D1, CDK4, p-AKT and p-4E-BP1 than the paired samples before palbociclib treatment. In conclusion, our findings suggest that overexpressed Cyclin D1 and CDK4 proteins lead to the resistance to CDK4/6 inhibitor and PI3K/mTOR inhibitors are able to restore the sensitivity to CDK4/6 inhibitors, which provides the biomarker and rationale for the combinational use of CDK4/6 inhibitors and PI3K/mTOR inhibitors after CDK4/6 inhibitor resistance in breast cancer. Citation Format: Qiang Liu, Zijie Cai, Jingru Wang, Yudong Li, Lok Lam Wong. Overexpressed cyclin D1 and CDK4 proteins are responsible for the resistance to CDK4/6 inhibitor in breast cancer that can be reversed by PI3K/mTOR inhibitors [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-04-01.

Abstract P5-10-06: Arginine- depleting enzyme, pegylated arginase, isolated from beef-liver tissue induces growth inhibition, apoptosis, cell cycle arrest and inhibits induced mammary tumors: A promising strategy for human breast cancer treatment in vitro and in vivo

Abstract Arginine auxotrophy occurs in certain tumor types, renders tumors vulnerable to treatment with arginine-degrading enzymes, and leads to their rapid demise. Arginine is a semi-essential amino acid but is essential for rapidly proliferating cells. Thus, Arginine deprivation can be exploited as a potential targeted therapy for the treatment of various cancers. Arginase, an enzyme of the urea cycle, converts L-arginine to L-ornithine, which is the precursor of polyamines that are essential components of cell proliferation. However, Arginine deprivation and Arginase clinical implications in human breast cancer have not been fully elucidated. The objective of the current study was to investigate the potential role of the Arginine degrading enzyme, Arginase on two breast cell lines that have different differentiation patterns, Luminal A breast cancer (T-47D), and TNBC (MDA-MB-231), respectively. Herein, Arginase was extracted and purified from the beef liver and its specific activity was 0.2 IU/mg protein yielded 49% compared to the crude beef liver Arginase. The purified Arginase is covalently bound via a succinamide propionic acid (SPA) linker to polyethylene glycol (PEG) of molecular weight 5,000 to enhance the half-life time compared to the native enzyme with a maximum specific activity between 37~55°C and pH 7~9. The potential influence of extracted Peg-Arginase was examined on cell proliferation, clonogenic assays, morphological alteration, and oncogenic signaling pathways of STAT3, β-catenin, and miR-23a. Our data showed that Peg-Arginase treatment displayed a significant inhibition in cell proliferation and viability in a dose-dependent manner in both breast cancer cell lines compared to control after 48h. Furthermore, mechanistic studies showed that Peg-Arginase induced apoptosis that was associated with cell morphological changes, histone release from fragmented DNA, caspase-3 activation, and cell cycle arrest at S and/or G2/M phases in a dose-. dependent manner after 48h. Moreover, Peg-Arginase down-regulated miR-23a, STAT3, β-catenin, cyclin D1, and mutant p53 levels as well as increased p21and Bax, pro-apoptotic signal, in both breast cancer cells as determined by qRT-PCR and western blot analysis. In vivo, the effects of native- and Peg-Arginase enzymes were examined on MNU-induced mammary tumors in Albino-Wister rats. Our data showed that injecting 400 IU of native- or peg- Arginase twice per week for two weeks significantly inhibited the growth of MNU-induced mammary tumors by 58.8±4.1 % for peg-Arginase compared to 19.7±3.5 % for native-Arginase. Furthermore, the results also revealed a significant reduction in activities of ALT, AST, and ALP in the Peg-Arginase group compared to MNU untreated or native-Arginase groups, suggesting that Peg-Arginase may eliminate the hepatotoxic effect of the MNU. Furthermore, Peg-Arginase treatment reduced the localization and expression of oncogenic β-catenin in the nucleus compared to MNU-untreated and native-Arginase groups. All in all, our results suggested that the arginine-depleting enzyme, Peg-Arginase, exerted an anticancer effect against both luminal A and triple-negative breast cancer cell lines, through cell proliferation inhibition and apoptosis induction, which might provide a promising alternative strategy targeting oncometabolite in human breast cancer. Citation Format: Islam Yahiya, Asmaa Ali, Omnia Ali, Ahmed Sultan. Arginine- depleting enzyme, pegylated arginase, isolated from beef-liver tissue induces growth inhibition, apoptosis, cell cycle arrest and inhibits induced mammary tumors: A promising strategy for human breast cancer treatment in vitro and in vivo [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-10-06.

MiR-597-5p suppresses the progression of hepatocellular carcinoma via targeting transcriptional enhancer associate domain transcription factor 1 (TEAD1)

Hepatocellular carcinoma (HCC) is the most common primary liver cancer with high incidence and mortality. MiR-597-5p is downregulated in tumor tissues of HCC compared with non-tumor tissues. However, its role in HCC is still unknown. This study aims to assess the function of miR-597-5p in HCC development and investigate the underlying mechanism. To perform gain- and loss-of-function studies, SK-HEP-1 cells and Huh-7 cells were transfected with miR-597-5p mimics and inhibitor, respectively. MiR-597-5p markedly reduced the cell viability and the expression of Ki-67 in HCC cells. MiR-597-5p also repressed the cell cycle progression of HCC cells and the protein levels of cyclin D1 and CDK2. Moreover, miR597-5p inhibited the migration and invasion of HCC cells and decreased MMP2 and MMP9 levels. Transcriptional enhancer associate domain transcription factor 1 (TEAD1) was identified as a target of miR-597-5p by luciferase reporter assay. TEAD1 and its downstream target genes, CTGF and CYR61, were downregulated by miR-597-5p in HCC cells. Furthermore, miR-597-5p was demonstrated to function in HCC progression by targeting TEAD1 via TEAD1 expression gain and loss. Our study demonstrates that miR-597-5p represses the proliferation, migration, and invasion of HCC cells through targeting TEAD1, which provides a therapeutic target for HCC treatment.

Evaluation of anti-cancer potential of Excoecaria\xa0agallocha (L.) leaf extract on human cervical cancer (SiHa) cell line and assessing the underlying mechanism of action

BackgroundThe incidence of cervical cancer is increasing at an alarming rate in many countries and presently, it is the most common form of malignant cancer being reported among women in India. Development of novel approach for cervical cancer therapy, sparing healthy normal cells overcoming the limitations of prevailing therapies is of prime importance. Mangroves constitute a significant repository of medicinally important plants. Thus, in this study, we aimed to determine the anticancer activity of the mangrove Excoecaria agallocha L. leaf extracts on human cervical cancer (SiHa HPV 16+) cell line with subsequent characterization of the bioactive compounds conferring the anticancer activity and studying the probable underlying mechanism of action of the purified plant extract.ResultsThe plant extract was subjected to silica gel column chromatography and the fractions obtained were analyzed for cytotoxic activity against SiHa cells by MTT assay. One out of the three eluted fractions exhibited selective toxicity against SiHa cells with an IC50 value of 15.538 ± 0.577 µg/mL, while it had no cytotoxic effect on normal healthy human peripheral blood mononuclear cells. High-resolution liquid chromatography mass spectroscopy, coupled to electron spray ionization and diode array detection analysis, led to the structure elucidation and identification of a few pharmacologically important compounds, with Bergenin being present in the highest abundance. Fluorescence microscopy results revealed that the plant extract fraction induced LC3 puncta formation, in EGFP- SiHa cells indicating the onset of autophagy, with simultaneous stimulation of mitophagy. The plant extract also inhibited proliferation of the SiHa-smac-mCherry cells by second mitochondria-derived activator of caspase (SMAC)—induced cytochrome c dependent apoptosis, that was further confirmed with Caspase-3 activation by colorimetric assay. The GFP-dgn in SiHa cells was remarkably protected from proteasomal degradation that might upregulate the survivability of the cells significantly. Flow cytometry followed by Western blot analysis further asserted the ability of the plant extract fraction to cause cell cycle arrest of SiHa cells in the G2/M phase by significantly reducing protein expression levels of cyclin B1 and D1, decreasing Cdc2 level and simultaneously increasing p21 and p53 levels.ConclusionIt could be inferred that the aqueous extract of E. agallocha successfully decreased the proliferation of SiHa cervical cancer cells through induction of autophagy and apoptosis in a concerted manner, with simultaneous stimulation of mitophagy and G2/M phase cell cycle arrest, hinting at Bergenin being the major compound conferring the anti-cancer activity of the plant extract. Thus, isolation of the identified bioactive compounds from E. agallocha and their subsequent purification for drug development might serve as a novel medicinal approach for the treatment of cervical cancer in conjugation with existing therapeutic methods.

Epigenetic network of EZH2/SFRP1/Wnt in the epithelial-mesenchymal transition of laryngeal carcinoma cells.

Enhancer of Zeste Homologue 2 (EZH2) as a histone methyltransferase epigenetically regulates laryngeal carcinoma (LGC) progression. The present study sought to explore the role and mechanism of EZH2 in the epithelial-mesenchymal transition (EMT) of LGC cells. Expressions of EZH2, secreted frizzled-related protein 1 (SFRP1), and trimethylation of lysine 27 on histone H3 (H3K27me3) in LGC tissues or cells were detected via reverse transcription quantitative polymerase chain reaction (qRT-PCR) and western blotting. Upon transfection of si-EZH2, si-SFRP1, oe-SFRP1, or H3K27me3 upregulation, cell viability was assessed via cell counting kit-8, protein levels of E-cadherin, N-cadherin, β-catenin, c-Myc, and Cyclin D1 were determined via western blotting, and Vimentin expression was determined via immunofluorescence. The enrichment level of H3K27me3 in the SFRP1 promoter was measured via chromatin immunoprecipitation-PCR. EZH2 was highly expressed in LGC tissues and cells. Silencing EZH2 repelled the EMT of LGC cells. Mechanically, EZH2 upregulated H3K27me3 in the SFRP1 promotor to inhibit SFRP1 expression, and SFRP1 overexpression inactivated the Wnt pathway. H3K27me3 upregulation or SFRP1 downregulation reversed the inhibition of silencing EZH2 in the EMT of LGC cells. Overall, EZH2 upregulated H3K27me3 in the SFRP1 promoter to inhibit SFRP1 expression and activate the Wnt pathway, thereby facilitating the EMT of LGC cells.

THERAPEUTIC PROFILING OF NANO ENCAPSULATED DIOSGENIN VIA ATTENUATING HORMONAL STATUS, CELL PROLIFERATION, INFLAMMATORY RESPONSES, AND APOPTOSIS IN AN ANIMAL MODEL OF MAMMARY ONCOGENESIS

Objective: The central motive of this study is to explore the therapeutic impact of Diosgenin encapsulated Chitosan nanoparticles (DG@CS-NP) on mammary carcinogenesis in female Sprague Dawley rats via modulating hormonal status, cell proliferation, inflammatory responses, and Apoptosis. Methods: 7,12-dimethylbenz(a)anthracene (DMBA) was administered subcutaneously near the mammary gland (25 mg/kg b. wt) to provoke mammary tumor in female Sprague Dawley rats. Following the progress of a tumor, DMBA-induced tumor-bearing rats were medicated orally with 5 mg/kg b. wt of DG@CS-NP. Consequently, the expression of ER, PR, PCNA, Cyclin D1, NF-κB, TNF-α, Bcl-2, Caspases-3, and p53 in experimental rats were revealed via architectural immunohistochemistry. Further, Diosgenin interactions with these proteins were evidently confirmed by molecular docking analysis. Results: As a result, we noticed diminished levels of ER, PR, PCNA, Cyclin D1, NF-κB, TNF-α, and Bcl-2 expressions in DG@CS-NP medicated rats as well as with elevated levels of Caspases-3 and p53 expressions. In DMBA rats, the expressions were vice versa. Additionally, molecular docking analyses support these outcomes by highlighting the strong interaction between Diosgenin and breast cancer targets. Conclusion: These reports prove that DG@CS-NP imposes its therapeutic impact by hormonal adjustments, downregulating proteins involved in inflammation and cellular proliferation, and thereby promotes apoptosis by impeding apoptotic inhibitors.

LPA2 protein is involved in photosystemII assembly in Chlamydomonas reinhardtii.

SummaryPhotosynthetic eukaryotes require the proper assembly of photosystem II (PSII) in order to strip electrons from water and fuel carbon fixation reactions. In Arabidopsis thaliana, one of the PSII subunits (CP43/PsbC) was suggested to be assembled into the PSII complex via its interaction with an auxiliary protein called Low PSII Accumulation 2 (LPA2). However, the original articles describing the role of LPA2 in PSII assembly have been retracted. To investigate the function of LPA2 in the model organism for green algae, Chlamydomonas reinhardtii, we generated knockout lpa2 mutants by using the CRISPR‐Cas9 target‐specific genome editing system. Biochemical analyses revealed the thylakoidal localization of LPA2 protein in the wild type (WT), whereas lpa2 mutants were characterized by a drastic reduction in the levels of D1, D2, CP47 and CP43 proteins. Consequently, reduced PSII supercomplex accumulation, chlorophyll content per cell, PSII quantum yield and photosynthetic oxygen evolution were measured in the lpa2 mutants, leading to the almost complete impairment of photoautotrophic growth. Pulse‐chase experiments demonstrated that the absence of LPA2 protein caused reduced PSII assembly and reduced PSII turnover. Taken together, our data indicate that, in C. reinhardtii, LPA2 is required for PSII assembly and proper function.

Aralia armata (Wall.) Seem Improves Intimal Hyperplasia after Vascular Injury by Downregulating the Wnt3α/Dvl-1/β-Catenin Pathway.

The aim of the study is to examine the mechanism of Aralia armata (Wall.) Seem (AAS) in improving intimal hyperplasia after vascular injury in rats. Rats with femoral artery injury were randomly divided into three groups: the model group, AAS low-dose group (40 mg/kg), and AAS high-dose group (80 mg/kg). The sham operation group was used as a control group. HE staining was used to observe the changes in femoral artery vessels. Immunohistochemistry was adopted to detect α-SMA, PCNA, GSK-3β, and β-catenin proteins in femoral artery tissue. The CCK-8 test and wound healing assay were employed to analyze the effect of AAS on proliferation and migration of vascular smooth muscle cells (VSMCs) cultured in vitro. Western blotting (WB) and polymerase chain reaction (PCR) assays were used to evaluate the molecular mechanism. AAS reduced the stenosis of blood vessels and the protein expressions of α-SMA, PCNA, GSK-3β, and β-catenin compared to the model group. In addition, AAS (0-15 μg/mL) effectively inhibited the proliferation and migration of VSMCs. Moreover, the results of WB and PCR showed that AAS could inhibit the activation of β-catenin induced by 15% FBS and significantly decrease the expression levels of Wnt3α, Dvl-1, GSK-3β, β-catenin, and cyclin D1 in the upstream and downstream of the pathway. AAS could effectively inhibit the proliferation and migration of neointima after vascular injury in rats by regulating the Wnt/β-catenin signaling pathway.

Effects of Xinwei granule on expression levels of cyclin D1 and its upstream genes in gastric intraepithelial neoplasia tissues

Purpose: To explore the effects of Xinwei granule (XWG) on low-grade gastric intraepithelial neoplasia (LGIN) and the underlying mechanisms.
 Methods: To establish LGIN model, Wistar rats were treated with N-methyl-N'-nitrosoguanidine for 3 months. LGIN model rats were randomly grouped into five groups (n = 15), viz, negative control (NC), normal saline (NS) group, Xinwei granule (XWG) group, Weifuchun tablet (WFCT) group, and vatacoenayme tablet (VT) group. Normal rats (n = 17) served as negative control. Histological evaluation of gastric mucosa was undertaken using hematoxylin and eosin staining. Quantitative realtime polymerase chain reaction (qRT-PCR), western blot, and immunohistochemical assays were performed to determine mRNA expressions, protein expression, and the distribution of cyclin D1, kruppel-like factor 4 (KLF4), and p21-WAF1-CIP1, respectively.
 Results: Compared with LGIN group, the body weight of the rats increased in XWG, WFCT, and VT groups. The pathological characteristics of LGIN group were alleviated by XWG, WFCT and VT treatments. The positive expression of cyclin D1 was enhanced in LGIN group, but reduced in XWG, WFCT and VT groups. The expression levels of KLF4 and p21-WAF1-CIP1, upstream regulators of cyclin D1 reduced in LGIN groups. However, administration of XWG, WFCT and VT strengthened the expressions of KLF4 and p21-WAF1-CIP1. More importantly, the protective effects of XWG against LGIN were superior to those of WFCT and VT.
 Conclusion: Xinwei granules alleviate LGIN in vivo by inhibiting cyclin D1 expression and enhancing KLF4 and p21-WAF1-CIP1 expression.