Cyclin D1 Protein Research Articles

Association of PIWI-interacting RNA-23210 with tumor metastasis in esophageal squamous cell carcinoma.

e16060 Background: his study aimed to analyze the expression, clinical significance of PIWI-interacting RNA-23210 (piRNA-23210) in esophageal squamous cell carcinoma (ESCC) and the biological effect in its cell line by piRNA-23210 overexpression. Methods: Quantitative real-time RT-PCR (qRT-PCR) was used to analyze piRNA-23210 expression in 60 cases of ESCC and 60 cases of normal tissues to study the relationship between piRNA-23210 expression and clinical factors. Recombinant lentiviral vector was constructed to overexpress piRNA-23210 and then infect ESCC TE-1 cell line. qRT-PCR was used to detect the level of piRNA-23210. 3-[4,5-dimethylthiazol -2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay, cell apoptosis, cell cycle, and migration and invasion assays were also conducted as to the influence of the upregulated expression of piRNA-23210 that might be found on TE-1 cells biological effect. Results: The level of piRNA-23210 expression was found to be significantly lower in ESCC tissue than normal tissues (P < 0.05). Decreased expression of piRNA-23210 was significantly correlated with lymph node metastasis, clinic stage, and histological grade of patients with ESCC (P < 0.05). Meanwhile, loss of piRNA-23210 expression correlated significantly with poor overall survival time by Kaplan–Meier analysis (P < 0.05). The result of biological function show that TE-1 cell transfected piRNA-23210 had a lower survival fraction; higher percentage of the G0/G1 phases; higher cell apoptosis; significant decrease in migration and invasion; and lower cyclin D1, Bcl-2, and MMP-7 protein expression compared with TE-1 cell untransfected piRNA-23210 (P < 0.05). Conclusions: piRNA-23210 expression decreased in ESCC and correlated significantly with lymph node metastasis, clinical stage, poor overall survival, cell proliferation, cell cycles, cell apoptosis and migration and invasion in ESCC cell by regulating cyclinD1, Bcl-2, and MMP-7 expression, suggesting that piRNA-23210 may play important roles as a negative regulator to ESCC cell.

Inhibiting the Expression of Cyclin D1 Protein to Reduce the HBVDNA Level in CHB patients

HBV virus is now affecting over 400 million people worldwide. Chronic Hepatitis B (CHB) is one of the significant clinical symptoms of HBV infection. Previous studies have reported that antiviral therapy is a practical method for patients. However, the current treatment has reached a bottleneck due to economic burden and drug resistance. As a practical component in ligustrum lucidum, ursolic acid has been found to have an antiviral effect. This study focuses on one particular mechanism of its effect. It tests its function in reducing the expression of Cyclin D1 protein to provide a possible reason for its ability to inhibit cell proliferation.

Sanwei DouKou Decoction ameliorate Alzheimer disease by increasing endogenous neural stem cells proliferation through the Wnt/β-catenin signalling pathway.

Sanwei DouKou decoction (SDKD) is a traditional Chinese medicine (TCM) prescription derived from the Tibetan medical book "Si Bu Yi Dian" and is clinically used for the treatment of Alzheimer's disease (AD). However, the potential mechanism of SDKD treatment for AD remains elusive. This study aims to explore the potential mechanism by which SDKD alleviates AD. Extracts of SDKD were identified with Gas chromatograph-mass spectrometer (GC-MS). 5×FAD mice were treated with SDKD for 8 weeks. The efficacy of SDKD against AD was evaluated by in-vivo experiments. Morris water maze and contextual fear conditioning tests were used to detect the learning and memory ability of mice. Hematoxylin-eosin staining (H&E) staining was used to observe the pathological changes of brain tissue. Immunohistochemistry was used to detect the positive expression of Nestin in hippocampus. In in-vitro experiments, the Cell Counting Kit 8 (CCK-8) technique was used to detect cell viability, the proliferation of neural stem cells was detected by immunofluorescence staining, the intracellular protein expression was detected by Western Blot. The results of this study suggested that SDKD may ameliorate AD. SDKD significantly shortened the escape latency of mice in the Morris water maze experiment, increased the number of times the mice crossed the target quadrant, and prolonged freezing time in the contextual fear memory experiment. SDKD also improved neuronal pathology in the hippocampus, decreased neuronal loss, and increased Nestin protein levels. Furthermore, in in-vitro experiments, SDKD could significantly increase Neural stem cells (NSCs) viability, promoted NSCs proliferation, and also effectively activated the Wnt/β-catenin signalling pathway, increased Wnt family member 3A (Wnt3a), β-catenin and CyclinD1 protein levels, activated the NSCs proliferation pathways in AD model mouse brain tissue. The present study demonstrated that sanwei doukou decoction can ameliorate AD by increasing endogenous neural stem cells proliferation through the Wnt/β-catenin signalling pathway. Our observations justify the traditional use of SDKD for a treatment of AD in nervous system.

Highland barley β-glucan boosted intestinal epithelial regeneration via cell cycle and autophagy

Cell cycle progression and autophagy take a crucial part in stimulating the regeneration of gut epithelial which definitely contributes to maintain the integrity of the intestinal barrier. Our previous research demonstrated that highland barley β-glucan (HBBG) significantly mitigated the damaged gastric mucosal and intestinal barrier of colitis mice, whenas the mechanism remains indistinct. In this study, we found that HBBG remitted the increase of disease activity index, the shortening of colon length and the damage of intestinal epithelial structure in mice with dextran sulphate sodium induced colitis. Furthermore, HBBG observably boosted gut epithelial regeneration on account of PCNA+ transit amplifying cells, Ki67+ crypt base columnar cells and goblet cells. Interestingly, HBBG relieved the apoptosis and suppressive proliferation of IEC-6 cells induced by lipopolysaccharide. The results of immunohistochemistry and flow cytometry demonstrated that HBBG promoted the cell cycle progression. Likewise, HBBG increased cell cycle related proteins CyclinD1 and CDK4 in IEC-6 cells. In the meanwhile, HBBG dramatically enhanced the autophagy. Excitingly, HBBG augmented the nuclear translocation of TFEB and phosphorylation of AMPK, which hinted HBBG activated autophagy via AMPK-TFEB signal. Expectantly, the treatment of 3-methyladenine, an autophagy inhibitor, inverted the promoted proliferation in IEC-6 cells caused by HBBG. Thereout, we presumed that HBBG accelerated the regeneration of intestinal epithelial by means of promoting cell cycle progression and activating the autophagy.

EZH2 activates Wnt/β-catenin signaling in human uterine fibroids, which is inhibited by the natural compound methyl jasmonate

To investigate the link between EZH2 and Wnt/β-catenin signaling and its role in uterine fibroids (UFs) pathogenesis and explore the potential effect of natural compound methyl jasmonate (MJ) against UFs. EZH2 overexpression or inhibition was achieved in human uterine leiomyoma (HuLM) cells using EZH2-expressing adenovirus or chemical EZH2 inhibitor (DZNep), respectively. The HuLM and normal uterine smooth muscle cells were treated with 0.1-3 mM of MJ, and several experiments were employed. Laboratory study. None. Methyl jasmonate. Protein expression of EZH2, β-catenin, and proliferating cell nuclear antigen (PCNA) was measured by Western blot as well as gene expression alterations of Wnt ligands (Wnt5A, Wnt5b, and Wnt9A), WISP1, CTNNB1, and its responsive gene PITX2 using quantitative real-time polymerase chain reaction. The protein and ribonucleic acid (RNA) levels of several markers were measured in MJ-treated or untreated HuLM cells, including EZH2 and β-catenin, extracellular matrix markers collagen type 1 (COL1A1) and fibronectin (FN), proliferation markers cyclin D1 (CCND1) and PCNA, tumor suppressor marker p21, and apoptotic markers (BAX, cytochrome c, and cleaved caspase 3). EZH2 overexpression significantly increased the gene expression of several Wnt ligands (PITX2, WISP1, WNT5A, WNT5B, and WNT9A), which increased nuclear translocation of β-catenin and PCNA and eventually HuLM cell proliferation. EZH2 inhibition blocked Wnt/β-catenin signaling activation where the aforementioned genes significantly decreased as well as PCNA, cyclin D1, and PITX2 protein expression compared with those in untreated HuLM. Methyl jasmonate showed a potent antiproliferative effect on HuLM cells in a dose- and time-dependent manner. Interestingly, the dose range (0.1-0.5 mM) showed a selective growth inhibitory effect on HuLM cells, not on normal uterine smooth muscle cells. Methyl jasmonate treatment at 0.5 mM for 24 hours significantly decreased both protein and RNA levels of EZH2, β-catenin, COL1A1, FN, CCND1, PCNA, WISP1, and PITX2 but increased the protein levels of p21, BAX, cytochrome, c and cleaved caspase 3 compared with untreated HuLM. Methyl jasmonate-treated cells exhibited down-regulation in the RNA expression of 36 genes, including CTNNB1, CCND1, Wnt5A, Wnt5B, and Wnt9A, and up-regulation in the expression of 34 genes, including Wnt antagonist genes WIF1, PRICKlE1, and DKK1 compared with control, confirming the quantitative real-time polymerase chain reaction results. Our studies provide a novel link between EZH2 and the Wnt/β-catenin signaling pathway in UFs. Targeting EZH2 with MJ interferes with the activation of wnt/β-catenin signaling in our model. Methyl jasmonate may offer a promising therapeutic option as a nonhormonal and cost-effective treatment against UFs with favorable clinical utility, pending proven safe and efficient in human clinical trials.

Angelica sinensis polysaccharides alleviate the oxidative burden on hematopoietic cells by restoring 5-fluorouracil-induced oxidative damage in perivascular mesenchymal progenitor cells

Context 5-Fluorouracil (5-FU)-injured stromal cells may cause chronic bone marrow suppression; however, the underlying mechanism remains unclear. Angelica sinensis polysaccharide (ASP), the main biologically active ingredient of the Chinese herb, Angelica sinensis (Oliv.) Diels (Apiaceae), may enrich the blood and promote antioxidation. Objective This study investigated the protective antioxidative effects of ASP on perivascular mesenchymal progenitors (PMPs) and their interactions with hematopoietic cells. Materials and methods PMPs were dissociated from C57BL/6 mouse femur and tibia and were subsequently divided into the control, ASP (0.1 g/L), 5-FU (0.025 g/L), and 5-FU + ASP (pre-treatment with 0.1 g/L ASP for 6 h, together with 0.025 g/L 5-FU) then cultured for 48 h. Hematopoietic cells were co-cultured on these feeder layers for 24 h. Cell proliferation, senescence, apoptosis, and oxidative indices were detected, along with stromal osteogenic and adipogenic differentiation potentials. Intercellular and intracellular signaling was analyzed by real-time quantitative reverse transcription polymerase chain reaction and Western blotting. Results ASP ameliorated the reactive oxygen species production/scavenge balance in PMPs; improved osteogenic differentiation; increased SCF, CXCL12, VLA-4/VCAM-1, ICAM-1/LFA1, and TPO/MPL, Ang-1/Tie-2 gene expression. Further, the ASP-treated feeder layer alleviated hematopoietic cells senescence (from 21.9 ± 1.47 to 12.1 ± 1.13); decreased P53, P21, p-GSK-3β, β-catenin and cyclin-D1 protein expression, and increased glycogen synthase kinase (GSK)-3β protein expression in co-cultured hematopoietic cells. Discussion and conclusions ASP delayed oxidative stress-induced premature senescence of 5-FU-treated feeder co-cultured hematopoietic cells via down-regulation of overactivated Wnt/β-catenin signaling. These findings provide a new strategy for alleviating myelosuppressive stress.

Mir-629 Repressed LATS2 Expression and Promoted the Proliferation of Prostate Cancer Cells.

MicroRNAs (miRNAs) have been discovered to play critical role in regulating prostate cancer (PC) progression. The function role of miR-629 in tumor progression of PC has not been studied. Here, we found that miR-629 was markedly upregulated in PC as determined using the cancer genome atlas (TCGA) dataset, clinical tissues, and cell lines. Functional analysis (MTT assays, colony formation assays, soft agar growth assay and BrdU incorporation assay) indicated that overexpression of miR-629 was drastically promoted, while miR-629-in significantly suppressed cell proliferation. LATS2 was predicted as a direct target of miR-629 and was confirmed by western blot and dual luciferase assay. Through downregulation of large tumor suppressor 2 (LATS2) by overexpression of miR-629, the p21 mRNA and protein were decreased while the Cyclin D3 mRNA and protein were enhanced, suggesting promoting of cell proliferation process. Additionally, knockdown of LATS2 reversed the inhibitory effect by miR-629-in in PC. Our study indicated that miR-629 might serve as a new promising target for PC treatment.

Small peptide LINC00511-133aa encoded by LINC00511 regulates breast cancer cell invasion and stemness through the Wnt/β-catenin pathway

LINC00511 is an long non-coding RNA (lncRNA) of ncRNAs,This study aimed to investigate whether the lncRNA LINC00511 could encode a small peptide, LINC00511-133aa, and whether this peptide could promote breast cancer cell metastasis and stemness by activating the wnt/β-catenin pathway. The LINC00511-133aa coding sequence vector and control vector were transfected into MCF-7 and MDA-MB-231 breast cancer cells, with subsequent assessment of peptide expression using PCR, western blotting, and immunofluorescence assays. Cell proliferation, invasion, and apoptosis were evaluated using CCK8, apoptotic, wound healing, and transwell invasion assays, while the characteristic changes of tumor stem cells were detected through sphere-forming assay and western blot analyses of the stemness markers Oct4, Nanog, and SOX2. Results showed that LINC00511-133aa was indeed encoded by LINC00511 and promoted the invasiveness and stemness of breast cancer cells while limiting apoptosis by modulating the expression levels of wnt/β-catenin pathway-related proteins Bax, c-myc, and CyclinD1, as well as facilitating β-catenin protein entry into the nucleus. This study provides evidence for the potential involvement of lncRNA LINC00511 and its peptide product in breast cancer progression via the regulation of the wnt/β-catenin pathway.

Tumor suppressive functions of hsa‑miR‑34a on cell cycle, migration and protective autophagy in bladder cancer.

Bladder cancer (BC) cells exhibit a high basal level of autophagy activity, which contributes to the development of a protective mechanism for cellular survival against current treatments. Hsa‑microRNA‑34a (miR‑34a) presents anti‑tumor function in several types of cancer. However, the functional mechanism of miR‑34a in regulating tumor aggressiveness and protective autophagy of BC remains largely unknown. First, transfected BC cells with miR‑34a mimic exhibited LC3‑II and p62 accumulation through immunofluorescence staining. It was demonstrated that syntaxin 17 (STX17), which is required for autophagosome‑lysosome fusion, was downregulated upon miR‑34a mimic treatment. Mechanistically, miR‑34a reduced the expression of STX17 proteins that directly bind on STX17 3'‑untranslated regions and thus suppressed STX17 mRNA translation to eventually inhibit protective autophagy in BC. Cell viability and colony formation assays revealed that overexpression of miR‑34a in BC cells enhances the chemosensitivity of cisplatin, doxorubicin, epirubicin and mitomycin C. Furthermore, miR‑34a inhibited cell proliferation and triggered G0/G1 cell cycle arrest by inhibiting cyclin D1 and cyclin E2 protein expression. Moreover, miR‑34a suppressed cell motility through the downregulation of epithelial‑mesenchymal transition. In summary, miR‑34a inhibits cell proliferation, motility and autophagy activity in BC, which can benefit BC treatment.

Galectin-3 Mediates Tumor Progression in Astrocytoma by Regulating Glycogen Synthase Kinase-3β Activity

Numerous studies have considered galectin-3 or Glycogen synthase kinase 3 beta (GSK3B) as a potential prognosis marker for various cancers. However, the correlation between the protein expression of galectin-3/GSK3B and the clinical parameters of astrocytoma has not been reported. This study aims to validate the correlation between the clinical outcomes and protein expression of galectin-3/GSK3B in astrocytoma. Immunohistochemistry staining was performed to detect galectin-3/GSK3B protein expression in patients with astrocytoma. The Chi-square test, Kaplan-Meier evaluation, and Cox regression analysis were used to determine the correlation between clinical parameters and galectin-3/GSK3B expression. Cell proliferation, invasion, and migration were compared between a non-siRNA group and a galectin-3/GSK3B siRNA group. Protein expression in galectin-3 or GSK3B siRNA-treated cells was evaluated using western blotting. Galectin-3 and GSK3B protein expression were significantly positively correlated with the World Health Organization (WHO) astrocytoma grade and overall survival time. Multivariate analysis revealed that WHO grade, galectin-3 expression, and GSK3B expression were independent prognostic factors for astrocytoma. Galectin-3 or GSK3B downregulation induced apoptosis and decreased cell numbers, migration, and invasion. siRNA-mediated gene silencing of galectin-3 resulted in the downregulation of Ki-67, cyclin D1, VEGF, GSK3B, p-GSK3B Ser9 (p-GSK3B S9), and β-catenin. In contrast, GSK3B knockdown only decreased Ki-67, VEGF, p-GSK3B S9, and β-catenin protein expression but did not affect cyclin D1 and galectin-3 protein expression. The siRNA results indicated that GSK3B is downstream of the galectin-3 gene. These data support that galectin-3 mediated tumor progression by upregulating GSK3B and β-catenin protein expression in glioblastoma. Therefore, galectin-3 and GSK3B are potential prognostic markers, and their genes may be considered to be anticancer targets for astrocytoma therapy.

Effect of Ethanolic Extract of Moringa oleifera, Lam. Roots on Expression of Cyclin D1 in Testicular Tissue of Metabolic Syndrome Induced Wistar Rats

Background: Metabolic syndrome (MS) impacts on inhibition process of spermatogenesis by damaging various components of cell cycle proteins. Cyclin D1 protein which plays role in regulating the cell cycle of spermatogenic cells is depleted due to the metabolic syndrome. Variation results of M. oleifera studies on fertility are interesting for further research. This study aimed to determine the effect of ethanolic extract of M. oleifera roots towards expression of testicular cyclin D1 of metabolic syndrome induced Wistar rats.Subjects and Method: This study was an experimental laboratory. Thirty subjects of male Wistar rats were randomly divided into 5 groups consisted of 6 rats, control group (K1), MS group (K2), and treatment groups (K3, K4, and K5) which were given doses of 150, 250, and 350 mg/kgBW, respectively for 28 days. Induction of MS was achieved by high-fat diet for 28 days and injection of streptozotocin-nicotinamide (STZ-NA) on day 25. Cyclin D1 data was calculated using Intensity Distribution Score (IDS) and analyzed using one-way ANOVA test followed by post hoc multiple comparisons with Tukey HSD. Simple liniar regression test conducted on cyclin D1 data of K2, K3, K4, and K5.Results: One-way ANOVA test showed that the administration of ethanolic extract M. oleifera Lam. roots with doses of 150, 250, and 350 mg/kgBW in 28 days increased cyclin D1 significantly (p= 0.000). There were significant differences between K1 and K2 (p= 0.000), K2 and K3 (p= 0.000), K2 and K4 (p= 0.000), K2 and K5 (P= 0.000). Liniar regression test showed significant (p= 0.000) and moderate effect (R2= 0.65) of doses of ethanolic extract of M. oleifera roots on the expression of cyclin D1.Conclusion: The administration of ethanolic extract Moringa oleifera Lam. roots with doses of 150, 250, and 350 mg/kgBW for 28 days increased cyclin D1 in testicular rats.

Abstract 6107: Novel roles for AMBRA1 in regulating DNA double-strand breaks

Abstract AMBRA1 (Activating Molecule in Beclin-1-Regulated Autophagy) is a tumor suppressor protein whose expression is downregulated in several human malignancies. AMBRA1 was recently found to function as a substrate receptor for the cullin-4-based ubiquitin ligase (CRL4AMBRA1) that promotes the ubiquitin-dependent proteolysis of D-type cyclins (cyclin D1, D2 and D3); this serves to guard against premature S-phase entry and replication stress, providing a potential mechanism for its tumor suppressive activity. Cyclin D1 protein, which is stabilized, along with cyclin D2 and D3, in AMBRA1-deficient cells, has been shown to promote homologous recombination (HR) repair of DNA double-strand breaks (DSBs), suggesting that AMBRA1 may be additionally involved in the repair of DSBs. Using our recently published CRISPR-Cas9-based dual fluorescent reporter assay system, we show that while the loss of AMBRA1 increased D-type cyclins and Rb hyperphosphorylation as expected, it did, surprisingly, inhibit HR-mediated repair and stimulated error-free non-homologous end-joining (NHEJ), and that both of these new activities are independent of cyclin D1 or RB1. High-throughput sequencing of DSB repair junctions confirm these results and shows significant depletion of indels with reduction in microhomology-mediated end-joining in AMBRA1-deficient cells, suggesting that AMBRA1 plays a role in promoting resection at DSBs. In support of this hypothesis, we found that the loss of AMBRA1 reduces RAD51 recruitment to DSBs in cells exposed to ionizing radiation. Consistent with its role in promoting HR, we found that AMBRA1-deficient cells are significantly more sensitive to PARP inhibitors compared to AMBRA1-proficient cells. In summary, we show that AMBRA1 is a novel regulator of DSBs, and that its downregulation in cancer cells and tumors render cancer cells susceptible to inhibition by PARP inhibitors. Citation Format: Yuning Jiang, Tarek Abbas. Novel roles for AMBRA1 in regulating DNA double-strand breaks. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6107.

Abstract 5255: Effect of STAT3 inhibitors, TTI-101 and SH5-07, against bladder cancer in preclinical 3D tumor models

Abstract Bladder cancer (BC) is a lethal genitourinary malignancy associated with frequent recurrence and poor survival due to metastatic potential. Identification of key cancer cell signaling networks and developing promising agents is critical for effectively inhibiting tumor growth and progression. In many cancers, including bladder cancer (BC), signal transducer and activator of transcription 3 (STAT3) has emerged as an important molecular pathway due to its role in promoting proliferation, invasion, and chemoresistance. Thus, developing STAT3 targeting, orally bioavailable small molecule inhibitors may be helpful for the prevention of BC progression and improving the survival rate of patients with muscle invasive BC. Monolayer culture has limitations for drug testing. Therefore, spheroid and organoid culture are used extensively as they may mimic in-vivo drug response more accurately. The aim of our study is to examine the preclinical anticancer efficacy of STAT3 inhibitors [TTI-101 (C188-9) and SH5-07] in 3D (spheroid and tumoroid) invitro models of BC. We optimized the spheroid growth using various BC cell lines [human (J82), rat (NBT-II), and mouse (MB49) BC cells]. Similarly, tumoroids from rat (BBN-induced bladder tumors) and transgenic mice (UPII-SV40T) bladder tumors were developed. These spheroids and tumoroids were treated with various concentrations (0 - 50 μM range) of STAT3 inhibitors and evaluated for their viability [Calcein AM (CA) and EtBr staining], ATP production (CellTiter-Glo™ 3D), and ROS production (MitoSOXTM). Effect of drug treatment on biomarkers of cell proliferation, apoptosis, stemness, STAT3 signaling, and immune modulation was determined using western blotting and immunofluorescence. Treatment with TTI-101 (0 - 50 μM or SH5-07 (0 - 50 μM) for 144 hrs resulted in significant reduction in the spheroids size (39-45% smaller Vs untreated; p<0.0001), along with decreased ATP levels (20%-40%, p<0.05). MitoSOXTM staining suggested that STAT3 inhibitors treatment increased ROS production in BC cells. CA and EtBr staining revealed that TTI-101 and SH5-07 treatment resulted in increased cell death in BC spheroids compared to control. Decreased spheroids and organoids size also correlated with increased apoptotic marker (cleaved caspase-3) along with decreased cyclin D1, PCNA, and pSTAT3 protein expression. Drug treated BC spheroids/tumoroids also showed reduction in CD44 (BC stemness and invasion marker) and induction of cGAS-STING pathway (cGAS, STING, TBK1, and IRF3) in comparison to the control. These findings indicate that STAT3 inhibitors, TTI-101 and SH5-07, could inhibit bladder cancer by suppressing STAT3 pathway activation and therefore warrant further study in vivo. (Supported by P30 CA225520 and Kerley-Cade Endowed Chair) Citation Format: Surya P. Singh, Gopal Pathuri, Adam Asch, Brian Cholewa, Robert Shoemaker, Chinthalapally V. Rao, Venkateshwar Madka. Effect of STAT3 inhibitors, TTI-101 and SH5-07, against bladder cancer in preclinical 3D tumor models. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5255.

YAP regulates the liver size during the fasting-refeeding transition in mice.

Liver is the central hub regulating energy metabolism during feeding-fasting transition. Evidence suggests that fasting and refeeding induce dynamic changes in liver size, but the underlying mechanisms remain unclear. Yes-associated protein (YAP) is a key regulator of organ size. This study aims to explore the role of YAP in fasting- and refeeding-induced changes in liver size. Here, fasting significantly reduced liver size, which was recovered to the normal level after refeeding. Moreover, hepatocyte size was decreased and hepatocyte proliferation was inhibited after fasting. Conversely, refeeding promoted hepatocyte enlargement and proliferation compared to fasted state. Mechanistically, fasting or refeeding regulated the expression of YAP and its downstream targets, as well as the proliferation-related protein cyclin D1 (CCND1). Furthermore, fasting significantly reduced the liver size in AAV-control mice, which was mitigated in AAV Yap (5SA) mice. Yap overexpression also prevented the effect of fasting on hepatocyte size and proliferation. Besides, the recovery of liver size after refeeding was delayed in AAV Yap shRNA mice. Yap knockdown attenuated refeeding-induced hepatocyte enlargement and proliferation. In summary, this study demonstrated that YAP plays an important role in dynamic changes of liver size during fasting-refeeding transition, which provides new evidence for YAP in regulating liver size under energy stress.

Antitumor activity of Metformin through p53 and Cyclin D1 in the Urothelial Cell Carcinoma

Bladder cancer is considered as one of the main drivers of cancer related mortality in adult men. Data from Global Cancer Statistics 2018 (GLOBOCAN) showed that the bladder cancer was included among the Top 10 cancer incidence in worldwide. Meanwhile, metformin, an antidiabetic agent, is believed to be able to impede the varying cancer cells expansion. Many examinations had displayed that metformin interferes via the AMPK/mTOR axis pathway, thereby suppressing tumor growth. AMPK activation can also increase stromal cell survival through p53 activation. Metformin also disrupts the cell cycle by decreasing the cyclin D1 protein in cancer cells. The human cell line 5637 was treated with metformin 15 mM, examined for cyclin D1 and p53 by immunohistochemical staining and assessed for the viability of cancer cells. The Statistic test was utilized to make a comparison of tumor viabilities and other variables. No significant differences were found in the expression of wild type p53 and cyclin D1 but significant differences were observed in the viability between the control and metformin groups. We have proven in our study that the anti-tumor effect of metformin in reducing the viability of urothelial carcinoma tumor cells not only through p53 and cyclin D1.

LncRNA SNHG12 promotes proliferation and migration of hepatic progenitor cells via the Wnt/β-catenin pathway.

Hepatic progenitor cells (HPCs) play an important role in the treatment of chronic liver disease. To investigate the effect and mechanism of long noncoding RNAs/small nucleolar RNA host gene 12 (lncRNA SNHG12) on the proliferation and migration of the HPC cell line WB-F344. Hepatic progenitor cells were divided into a no-treatment group (sham), empty vector transfection of plasmid pcDNA3.1 (NC vector), pcDNA3.1-SNHG12 (SNHG12), negative short hairpin RNA (sh-NC), SNHG12 shRNA (sh-SNHG12), and pcDNA3.1-SNHG12+salinomycin intervention (SNHG12+salinomycin) groups. Cell proliferation, cell cycle and migration ability, as well as albumin (ALB), alpha-fetoprotein (AFP), â-catenin, cyclin D1, and c-Myc protein expression in each group were determined using Cell Counting Kit-8 (CCK-8), 5-ethynyl-2'-deoxyuridine (EdU), flow cytometry, transwell migration assays, enzyme-linked immunosorbent assay (ELISA), and western blot. The overexpression of lncRNA SNHG12 significantly upregulated proliferation, migration and cell cycle progression of WB-F344 cells. Furthermore, the overexpression of lncRNA SNHG12 increased the level of ALB, and the protein expression of â-catenin, cyclin D1 and c-Myc in the cell line, while decreasing the level of AFP. Conversely, the knockdown of lncRNA SNHG12 displayed the opposite effects. The inhibition of the Wnt/â-catenin signaling pathway with salinomycin significantly downregulated the â-catenin, cyclin D1 and c-Myc protein expression in WB-F344 cells. The lncRNA SNHG12 promotes the proliferation and migration of WB-F344 cells via activating the Wnt/â-catenin pathway.

The effects of the prognostic biomarker SAAL1 on cancer growth and its association with the immune microenvironment in lung adenocarcinoma

BackgroundInhibition of Serum Amyloid A-like 1 (SAAL1) expression could inhibit cancer progression and improve the prognosis of cancer patients. At present, the correlation between SAAL1 and lung adenocarcinoma (LAC) remains unclear. Therefore, this study surveyed the worth and pathway of SAAL1 in LAC progression and immunity.MethodsBioinformatics and immunohistochemistry were used to identify the SAAL1 expression in LAC. The roles of SAAL1 expression in the existence values of LAC patients were explored, and the nomograms were constructed. Clinical values of SAAL1 co-expressed genes were evaluated by COX regression, survival, and Receiver operating characteristic (ROC) analysis. EDU and western blotting methods were used to inquiry the functions and pathways of the SAAL1 in cell growths. The correlation between the SAAL1 level and immune microenvironment was visualized using correlation research.ResultsSAAL1 level was elevated in LAC tissues, and was observed in cancer tissues of dead patients. SAAL1 overexpression had something to do with shorter overall survival, progression-free interval, and disease-specific survival in LAC. The area under the curve of SAAL1 was 0.902 in normal tissues and cancer tissues. Inhibition of SAAL1 expression could inhibit cancer cell proliferation, which may be related to the decreased expression of cyclin D1 and Bcl-2 proteins. In LAC, SAAL1 level had something to do with stromal, immune, and estimate scores, and correlated with macrophages, T cells, Th2 cells, CD8 T cells, NK CD56dim cells, DC, eosinophils, NK CD56bright cells, pDC, iDC, cytotoxic cells, Tgd, aDC cells, B cells, Tcm, and TFH levels. SAAL1 overexpression had something to do with existence values and the immunity in LAC.ConclusionsInhibition of SAAL1 expression could regulate cancer growth via cyclin D1 and Bcl-2. SAAL1 is a promising prognostic biomarker in LAC patients.

Ethanolic Extract of Artemisia vulgaris Leaf Promotes Apoptotic Cell Death in Non-Small-Cell Lung Carcinoma A549 Cells through Inhibition of the Wnt Signaling Pathway.

The Wnt signaling pathway is reported to be associated with lung cancer progression, metastasis and drug resistance, and thus it is an important therapeutic target for lung cancer. Plants have been shown as reservoirs of multiple potential anticancer agents. In the present investigation, the ethanolic leaf extract of Artemisia vulgaris (AvL-EtOH) was initially analyzed by means of gas chromatography-mass spectrometry (GC-MS) to identify the important phytochemical constituents. The GC-MS analysis of AvL-EtOH exhibited 48 peaks of various secondary metabolites such as terpenoids, flavonoids, carbohydrates, coumarins, amino acids, steroids, proteins, phytosterols, and diterpenes. It was found that the treatment with increasing doses of AvL-EtOH suppressed the proliferation and migration of lung cancer cells. Furthermore, AvL-EtOH induced prominent nuclear alteration along with a reduction in mitochondrial membrane potential and increased ROS (reactive oxygen species) generation in lung cancer cells. Moreover, AvL-EtOH-treated cells exhibited increased apoptosis, demonstrated by the activation of caspase cascade. AvL-EtOH also induced downregulation of Wnt3 and β-catenin expression along with cell cycle protein cyclin D1. Thus, the results of our study elucidated the potential of bioactive components of Artemisia vulgaris in the therapeutic management of lung cancer cells.

Circ_0078607 increases platinum drug sensitivity via miR-196b-5p/GAS7 axis in ovarian cancer

ABSTRACT Platinum-based chemotherapy is one of the predominant strategies for treating ovarian cancer (OC), however, platinum resistance greatly influences the therapeutic effect. Circular RNAs (circRNAs) have been found to participate in the pathogenesis of platinum resistance. Our aim was to explore the involvement of circ_0078607 in OC cell cisplatin (DDP) resistance and its potential mechanisms. Circ_0078607, miR-196b-5p, and growth arrest-specific 7 (GAS7) levels were assessed by qPCR. Circ_0078607 stability was assessed by ribonuclease R digestion and actinomycin D treatment. Cell viability of various conic of DDP treatment was measured by CCK-8. The cell proliferation was determined by CCK-8 and colony formation assay. Western blotting was performed for determining GAS7, ABCB1, CyclinD1 and Bcl-2 protein levels. The direct binding between miR-196b-5p and circ_0078607 or GAS7 was validated by dual-luciferase reporter and RIP assay. DDP resistance in vivo was evaluated in nude mice. Immunohistochemistry staining for detecting Ki67 expression in xenograft tumours. Circ_0078607 and GAS7 was down-regulated, but miR-196b-5p was up-regulated in OC samples and DDP-resistant cells. Overexpression of circ_0078607 inhibited DDP resistance, cell growth and induced apoptosis in DDP-resistant OC cells. Mechanistically, circ_0078607 sequestered miR-196b-5p to up-regulate GAS7. MiR-196b-5p mimics reversed circ_0078607 or GAS7 overexpression-mediated enhanced sensitivity. Finally, circ_0078607 improved the sensitivity of DDP in vivo. Circ_0078607 attenuates DDP resistance via miR-196b-5p/GAS7 axis, which highlights the therapeutic potential of circ_0078607 to counter DDP resistance in OC.

Adipose Tissue-Mesenchymal Stem Cells Caused to Change the Methylation Status of hTERT Gene Promoter CpG Islands of Molt-4 Leukemia Cells as Cell-based Therapy.

DNA methylation was considered as prognostic information in some hematological malignancies. Previous studies have reported the in vitro and in vivo biology role of mesenchymal stem cells (MSCs) on leukemic cells. The aim of this study was to investigate the effect of MSCs on the promoter methylation status of hTERT as a catalytic subunit of telomerase enzyme. In the experimental study, the Molt-4 leukemic cells were co-cultured with MSCs for 7 days. At the end of the co-culture period, the Molt-4 cells were collected, DNA and protein were extracted. Then methylation specific-PCR and western blotting were done for evaluating the hTERT gene promoter methylation status and cyclin D1 and hTERT protein expression, respectively. In the following, the flow cytometry was done for cell cycle distribution assay. It was found that MSCs resulted in a significant decrease in the cyclin D1 and hTERT protein expression levels. Also, MSCs caused changes in the methylation status of the CpG islands in the hTERT gene promoter region. The following results showed that MSCs caused a significant increase in the number of cells at G0/G1 phase and arrest the G0/G1 phase as well as decrease in the cell proliferation of Molt-4 cells. It is concluded that co-culture of MSCs with Molt-4 cells could be involved in changing the methylation status of hTERT gene promoter, cell cycle and hTERT protein expression; it could be potentially beneficial for further investigations regarding the cell transplantation and cell-based therapy.