P53 mRNA Levels Research Articles

Does walking during chemotherapy impact p16INK4a levels in women with early breast cancer.

Increased p16INK4a (p16) expression is directly related to cellular senescence and is a robust biomarker of aging in humans. Prior studies have shown that levels of p16 dramatically increase in breast cancer patients who have received adjuvant chemotherapy. This study investigated whether moderate physical activity during chemotherapy would attenuate the expected rise in p16 expression. Participants were women with Stage I-III breast cancer enrolled in a walking study for the duration of their chemotherapy (NCT02167932, NCT02328313, NCT03761706). Participants were asked to walk at least 30 min or 6200 steps/day following a structured walking program and to wear an activity tracker. p16 mRNA levels were measured in peripheral blood T-cells before chemotherapy initiation and at approximately 6 months after last chemotherapy treatment (mean 200 days, SD 40 days). In total, 141 participants met inclusion criteria and 10% (n=14) averaged > 6200 steps/day. There was no significant association of daily steps with change in p16 levels pre- to post-chemotherapy (Pearson correlation coefficient=0.11, p=0.17). After adjusting for age, stage, anthracycline-based chemotherapy, and baseline p16, the change in log2 p16 for each 1000 steps was estimated to be 0.03 (p=0.35). Most participants were sedentary prior to chemotherapy and achieved modest levels of physical activity during treatment. A self-guided walking program achieved only modest levels of physical activity and was unable to ameliorate chemotherapy-induced change in p16 levels in women undergoing chemotherapy for early-stage breast cancer. More structured and vigorous exercise programs should be tested for a more definitive exploration of their impact on post-chemotherapy p16 levels.

Low miR-222 expression in human visceral adipose tissue is associated with insulin resistance and PTEN and p53 mRNA levels.

The aim of this is study was to analyse the expression of miR-193b, miR-378, miR-Let7-d, and miR-222 in human visceral adipose tissue (VAT), as well as their association with obesity, insulin resistance (IR), and their role in the regulation of genes controlling adipose tissue homeostasis, including adipocytokines, the phosphatase and tension homologue (PTEN), and tumour protein 53 (p53). VAT was obtained from normal-weight (NW), overweight, and obese (OW/OB) subjects with and without IR. Stem-loop RT-qPCR was used to evaluate miRNA expression levels. miRTarBase 4.0, miRWalk, and DIANA-TarBase v8 were used for prediction of validated target gene of the miRNA analysed. A qPCR was used to evaluate PTEN, p53, leptin (LEP), and adiponectin (ADIPOQ) mRNA. miR-222 was lower in IR subjects, and miR-222 and miR-378 negatively correlated with HOMA-IR. PTEN and p53 are miR-222 direct targets according to databases. mRNA expression of PTEN and p53 was lower in OW/OB subjects with and without IR, compared to NW group and its levels positively associated with miR-222. Additionally, p53 and PTEN are positively associated with serum leptin levels. On the other hand, miR-193b and miR-378 negatively correlated with serum leptin but not with mRNA levels. Moreover, miR-Let-7d negatively correlated with serum adiponectin but not with adiponectin mRNA levels. Lower miR-222 levels are associated with IR, and PTEN and p53 expression; the implication of these genes in adipose tissue homeostasis needs more research.

FSH-inhibited autophagy protects against oxidative stress in goat Sertoli cells through p62-Nrf2 pathway

Oxidative stress is a common cause of male infertility. Sertoli cells are one of the target cells of oxidative injury, which leads to impaired testicular function. Follicle-stimulating hormone (FSH) is critical in Sertoli cell function. However, the role of FSH in the response of goat Sertoli cells to H2O2-induced oxidative stress has not been studied yet. To investigate this response, we established an oxidative stress model using goat Sertoli cells. FSH pretreatment significantly enhanced the decreased cell viability (p < 0.05) caused by oxidative injury and inhibited autophagic flux. FSH significantly increased p62 mRNA and protein levels (p < 0.01). Further investigations revealed that FSH also increased the expression level and nuclear translocation of Nrf2 in Sertoli cells (p < 0.01), which resulted in increased antioxidant enzyme activity (p < 0.05). In contrast, treatment with siNrf2 and sip62 abolished this protective effect of FSH. These findings suggest that FSH protects Sertoli cells against oxidative stress via the p62-Nrf2 pathway, and that p62 accumulation maintains persistent activation of Nrf2. Thus, p62 and Nrf2 are required for FSH-mediated protective role in H2O2-induced Sertoli cell injury. The findings reveal new mechanisms by which FSH protects against oxidative injury in goat Sertoli cells.

Indole-3-Carbinol Stabilizes p53 to Induce miR-34a, Which Targets LDHA to Block Aerobic Glycolysis in Liver Cancer Cells.

Certain cancer cells prefer aerobic glycolysis rather than oxidative phosphorylation for energy supply. Lactate dehydrogenase A (LDHA) catalyzes the reduction of pyruvate to lactate and regains NAD+ so that glycolysis is continued. As a pivotal enzyme to promote smooth glycolysis, LDHA plays an important role in carcinogenesis. Indole-3-carbinol (I3C) has displayed antitumor activity, but the exact mechanism remains to be identified. In this study, we treated liver cancer cells with I3C, performed colony formation and cell migration, measured the expression of glycolysis-related proteins, and predicted and validated LDHA-targeting miRNA from the databases. In addition, the mRNA and protein levels of p53, glycolysis-related genes and miRNAs that regulate glycolysis were detected after I3C and siRNA-p53 treatment alone or in combination. Next, the expression and colocalization of p53 and MDM2 in liver cancer cells were evaluated after I3C treatment, and the effect of I3C on p53 protein stability was examined. The results showed that I3C inhibited cell proliferation, migration, and the expression levels of glycolysis-related gene LDHAs. MiR-34a was predicted to target LDHA, and I3C downregulated its expression. Furthermore, the combined I3C and siRNA-p53 treatment demonstrated that I3C regulated the expression of LDHA via miR-34a in a p53-dependent manner. Finally, I3C inhibited MDM2 expression and its colocalization with p53 and stabilized p53 expression. In summary, I3C inhibited the degradation of p53 by MDM2 in liver cancer cells; stable p53 induced miR-34a, which targeted LDHA, a key enzyme for aerobic glycolysis, suggesting cancer metabolism is an important target for I3C in liver cancer cells.

Knockdown of Toe1 causes developmental arrest during the morula-to-blastocyst transition in mice

The target of EGR1 protein 1 (TOE1) is evolutionarily conserved from Caenorhabditis elegans to mammals, which plays a critical role in the maturation of a variety of small nuclear RNAs. Mutation in human TOE1 has been reported to cause pontocerebellar hypoplasia type 7, a severe neurodegenerative syndrome. However, the role of TOE1 in early embryonic development remains unclear. Herein, we found that Toe1 mRNA and protein were expressed in mouse preimplantation embryos. Silencing Toe1 by siRNA led to morula-to-blastocyst transition failure. This developmental arrest can be rescued by Toe1 mRNA microinjection. EdU incorporation assay showed a defect in blastomere proliferation within developmentally arrested embryos. Further studies revealed that Toe1 knockdown caused increased signals for γH2AX and micronuclei, indicative of sustained DNA damage. Moreover, mRNA levels of cell cycle inhibitor p21 were significantly upregulated in Toe1 knockdown embryos before developmental arrest. Together, these results suggest that TOE1 is indispensable for mouse early embryo development potentially through maintaining genomic integrity. Our findings provide further insight into the role of TOE1 in mouse preimplantation embryonic development.

Secoisolariciresinol diglucoside lignan concentrate of flaxseeds exhibits chemoprotective role in non-melanoma skin cancer through inhibition of CDK4 and upregulation of p53

Cyclin-dependent kinases (CDKs) serve as target for various cancers including skin cancer. Secoisolariciresinoldiglucoside (SDG) and lignans exert anticancer effect on colon cancer through inhibition of CDKs. However, reports of SDG lignan concentrate (SLC) of Linum usitatissimum (L.) on skin cancer are not available. Hence, in this study, we evaluated the effect of SLC of L. usitatissimum on skin cancer, and determined the mechanism of action. Cell viability studies were done using the A-375 cell line. Skin cancer was induced by dimethyl Benz(a) anthracene and croton oil in female balb/c mice. SLC (5%) was administered from the 7th to 16th week after which we evaluated serum and tissue parameters. The IC50 value of SLC was found to be 93.7 μg/mL on the A-375 cell line. Skin cancer control animals exhibited increased tumor volume and burden and an increase in non-specific serum markers and tissue markers. Treatment with SLC decreased tumor volume and burden, and serum and tissue markers. Histopathological studies also depicted protection with SLC treatment. Docking studies revealed that SDG exhibits a good binding score with CDK4. Skin cancer control mice showed significantly increased CDK4 mRNA and decreased p53 mRNA levels which were prevented by SLC treatment. SLC exhibited a chemopreventive effect in skin cancer depicted by a reduction in serum biomarker, oxidative stress, collagen levels, tumor volume, tumor burden, and histopathological studies. These effects are mediated through the inhibition of CDK4 and upregulation of p53.

التأثير التآزري المضاد للأورام للثيموكينون مع الريسفيراترول ضد خط خلايا سرطان الرئة البشرية

Introduction: Thymoquinone (TQ) and resveratrol are natural anticancer and chemo-preventive agents through mechanism(s) that are not fully understood. This study aimed to test the anticancer potential activity of the combination of TQ and Resveratrol, and their underlying mechanisms in the human lung cancer cell line, A549 cells. Materials and methods: The A549 cells were treated with the different concentrations (25, 50, 75, 100, or 200 μM) of TQ, resveratrol, and their combination for 24h. The antiproliferative activity against A549 cells was assessed using (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and Trypan blue exclusion assay. Apoptosis markers were assessed by using a real-time reverse transcriptase PCR Assay (rRT-PCR). Results : We found that the proliferation of A549 lung cancer cells was inhibited by TQ, and resveratrol and their combination in a concentration-dependent manner, during a defined period. The combination therapy also could inhibit the growth of A549 cells at 25 μM with a 50% inhibitory concentration (IC50) value of 37 ± 3.18μM. These combinations were significantly (P &lt; 0.001) elevated caspase-3, caspase-8, caspase-9, p53, p21, Fas, FasL, and Bax mRNA levels in treating A549 cells compared to the untreated cells. The anticancer effect of this combination is mediated by the activation of the extrinsic and the intrinsic pathways. Conclusion: all these results give a new promising combination with the enhanced anticancer effect.

Amygdalin as a chemoprotective agent in co-treatment with cisplatin.

Amygdalin is a naturally occurring glycoside used in traditional Chinese medicine and is known to have anti-cancer properties. Even though the anti-cancer properties of amygdalin are well known, its effect on normal cells has not been thoroughly investigated. The aim of the present study was to investigate a possible chemo-protective role of amygdalin against the cytotoxic effects of chemotherapy for normal human cells. Specifically, it was tested in combination with a strong chemotherapeutic drug cisplatin. Human non-tumorigenic MCF12F epithelial cell line, human fibroblasts cells, human breast cancer MCF7 and MDA-MB-231 cells were treated with cisplatin in a dose- and time-depended manner in the absence or presence of amygdalin. When MCF12F cells and fibroblasts underwent pre-treatment with amygdalin followed by cisplatin treatment (24 h amygdalin + 24 h cisplatin), the cell viability was increased (22%, p < 0.001) as indicated using MTT assay. As attested by flow cytometry, combination treatment was associated with decreased the percentage of late apoptotic cells compared with monotherapy (fold-change of decrease = 1.6 and 4.5 for 15 and 20 μΜ, respectively). Also, the proteins expression of PUMA, p53, phospho-p53 and Bax decreased, when a combination treatment was used vs. cisplatin alone, while the proapoptotic proteins Bcl-2 and Bcl-xL exhibited an increased tendency in the presence of amygdalin. Moreover, the levels of pro-apoptotic genes PUMA, p53, and BAX mRNA were significantly downregulated (∼83%, ∼66%, and ∼44%, respectively) vs. cisplatin alone, while the mRNA levels of anti-apoptotic genes BCl-2 and Bcl-XL were upregulated (∼44.5% and ∼51%, respectively), vs. cisplatin alone after 24 h of combination treatment. The study on the Combination index (CI) assay indicated that amygdalin could be possibly considered as an antagonist to cisplatin (2.2 and 2.3) for MCF12F and fibroblast cells, respectively. In contrast, for the breast cancer MCF7 and MDA-MB-231 cells, amygdalin and cisplatin indicated a synergistic effect (0.8 and 0.65), respectively. Our present findings suggest that amygdalin has chemo-modulatory effect when used in co-treatment with cisplatin and is able to protect normal breast cells as well as the fibroblasts during chemotherapy treatment, indicating a strong selective chemoprotective ability and may contribute to a better quality of life for cancer patients.

Design and synthesis of N-(1-(6-(substituted phenyl)-pyridazin-3-yl)-piperidine-3-yl)-amine derivatives as JMJD6 inhibitors

JMJD6 is a member of the JmjC domain-containing family and has been identified as a promising therapeutic target for treating estrogen-induced and triple-negative breast cancer. To develop novel anti-breast cancer agents, we synthesized a class of N-(1-(6-(substituted phenyl)-pyridazine-3-yl)-piperidine-3-yl)-amine derivatives as potential JMJD6 inhibitors. Among them, the anti-cancer compound A29 was an excellent JMJD6 binder (KD = 0.75 ± 0.08 μM). It could upregulate the mRNA and protein levels of p53 and its downstream effectors p21 and PUMA by inhibiting JMJD6. Besides, A29 displayed potent anti-proliferative activities against tested breast cancer cells by the induction of cell apoptosis and cell cycle arrest. Significantly, A29 also promoted a remarkable reduction in tumor growth, with a TGI value of 66.6% (50 mg/kg, i.p.). Taken together, our findings suggest that A29 is a potent JMJD6 inhibitor bearing a new scaffold acting as a promising drug candidate for the treatment of breast cancer.

Mechanism of Atractylodes macrocephala against Alzheimer's disease via regulating lysophagy based on LKB1-AMPK-TFEB pathway

Myloid beta(Aβ) is produced by cleavage of amyloid precursor protein(APP), which is a main reason for Alzheimer's disease(AD) occurrence and development. This study preliminarily investigated the mechanism of Atractylodes macrocephala(AM) against AD based on LKB1-AMPK-TFEB pathway. The effect of AM on memory ability of AD transgenic Caenorhabditis elegans CL2241 was detected, and then the APP plasmid was transiently transferred to mouse neuroblastoma(N2 a) cells in vitro. The mice were divided into the blank control group, APP group(model group), positive control group(100 μmol·L~(-1) rapamycin), and AM low-, medium-and high-dose groups(100, 200 and 300 μg·mL~(-1)). The content of Aβ_(1-42) in cell medium, the protein level of APP, the fluorescence intensity of APP, the transcriptional activity of transcription factor EB(TFEB), the activity of lysosomes in autophagy, and autophagy flux were determined by enzyme-linked immunosorbent assay(ELISA), Western blot, fluorescence microscope, luciferase reporter gene assay, RLuc-LC3 wt/RLuc-LC3 G120 A, and mRFP-GFP-LC3, respectively. The protein expression of TFEB, LC3Ⅱ, LC3Ⅰ, LAMP2, Beclin1, LKB1, p-AMPK and p-ACC was detected by Western blot. Immunofluorescence and reverse transcription-polymerase chain reaction(RT-PCR) were used to detect the fluorescence intensity of TFEB and the mRNA expression of TFEB and downstream target genes, respectively. The results showed that AM reduced the chemotactic index of transgenic C. elegans CL2241, and decreased the content of Aβ in the supernatant of cell culture medium at different concentrations. In addition, AM lowered the protein level of APP and the fluorescence intensity of APP in a dose-dependent manner. Transcriptional activity of TFEB and fluorescence intensity of mRFP-GFP-LC3 plasmid were enhanced after AM treatment, and the value of RLuc-LC3 wt/RLuc-LC3 G120 A was reduced. AM promoted the protein levels of TFEB, LAMP2 and Beclin1 at different concentrations, and increased the protein expression ratio of LC3Ⅱ/LC3Ⅰ in a dose-dependent manner. Immunofluorescence results revealed that AM improved the fluorescence intensity and nuclear expression of TFEB, and RT-PCR results indicated that AM of various concentrations elevated the mRNA expression of TFEB in APP transfected N2 a cells and promoted the transcription level of LAMP2 in a dose-dependent manner, and high-concentration AM also increased the mRNA levels of LC3 and P62. The protein levels of LKB1, p-AMPK and p-ACC were elevated by AM of different concentrations. In summary, AM regulating lysophagy and degrading APP are related to the activation of LKB1-AMPK-TFEB pathway.

Insulin-like growth factor binding protein 5 accelerate the senescence of periodontal ligament stem cells.

Evidences have showed stem cell mediated tissue regeneration is a promising method for the treatment of periodontitis. Insulin-like growth factor binding proteins-5 (IGFBP5) is a member of the insulin growth factor (IGFs) family and plays a regulatory role in cell proliferation and differentiation. Our previous study showed that IGFBP5 can promote osteogenic differentiation of periodontal ligament stem cells (PDLSCs) and enhance periodontal tissue regeneration mediated by PDLSCs. However, the function of IGFBP5 in the process of PDLSCs senescence remains unclear. The present study showed IGFBP5 mRNA level was highly expressed in passage-induced aged PDLSCs cells. IGFBP5 knockdown decreased the ratio of senescence associated β-galactosidase (SA-β-Gal) positive cells, enhanced the activity of TERT, and down-regulated the expression levels of P16, P21, P53 mRNA and protein. Overexpression of IGFBP5 increased the ratio of SA-β-Gal positive staining PDLSCs, decreased the activity of telomerase TERT, and up-regulated the expression levels of P16, P21, P53 mRNA and protein related to PDLSCs senescence. In conclusion, IGFBP5 can accelerate the senescence of PDLSCs, indicating the potential target for maintaining the "young state" of stem cells.

The role of p53-MDM2 signaling in missed abortion and possible pathogenesis.

Missed abortion is one of the common obstetrical and gynecological complications, angiogenesis is the most important factor in fetal and placental development. However, the definite etiology and pathogenesis are not fully understood. The mRNA levels of p53, MDM2, VEGF, and HIF-lα were detected in 60 villous samples of missed abortion patients and 64 healthy controls by quantitative reverse-transcription polymerase chain reaction (qRT-PCR). Immunohistochemistry was used to explore the expression and correlation of p53, MDM2, VEGF, and HIF-lα in villous tissues. Furthermore, we upregulated MDM2 expression in JEG-3 and BeWo cells under hypoxia, combined with Nutlin3, the cell cycle was determined using flow cytometry and the expressions of p53, MDM2, VEGF, and HIF-1α were determined by qRT-PCR and western blot. qRT-PCR demonstrated that the expressions of p53, MDM2, and HIF-1α were significantly increased and VEGF was decreased in missed abortion group compared with normal pregnancies. Correlation analysis found that p53 was positively correlated with MDM2 and HIF-1α, and negatively correlated with VEGF in missed abortion group. After administration of Nutlin3, overexpression of MDM2 could arrest cell cycle in G1 phase and reduce the proportion of S phase. The expression of p53 and MDM2 of JEG-3 cells and BeWo cells which transfected with pcDNA3.1-MDM2 plasmid were markedly increased after Nutlin3 addition under hypoxic conditions, while the expression of VEGF and HIF-1α decreased. Our data indicated that during the development of villi in early pregnancy, p53-MDM2 signaling regulate cell cycle and angiogenesis through interaction with HIF-1α and VEGF, which may be a crucial factor affecting pregnancy outcomes.

Cadmium exposure induces autophagy via PLC-IP3 -IP3 R signaling pathway in duck renal tubular epithelial cells.

Cadmium (Cd) is detrimental to animals, but nephrotoxic effects of Cd on duck have not been fully elucidated. To evaluate the impacts of Cd on Ca homeostasis and autophagy via PLC-IP3 -IP3 R pathway, primary duck renal tubular epithelial cells were exposed to 2.5μM and 5.0μM Cd, and combination of 5.0μM Cd and 10.0μM 2-APB or 0.125 μM U-73122 for 12 h (U-73122 pretreated for 1h). These results evidenced that Cd induced [Ca2+ ]c overload mainly came from intracellular Ca store. Cd caused [Ca2+ ]mit and [Ca2+ ]c overload with [Ca2+ ]ER decrease, elevated Ca homeostasis related factors (GRP78, GRP94, CRT, CaN, CaMKII, and CaMKKβ) expression, PLC and IP3 activities and IP3 R expression, but subcellular Ca2+ redistribution was reversed by 2-APB. PLC inhibitor U-73122 dramatically relieved the changes of the above indicators induced by Cd. Additionally, U-73122 obviously reduced the number of autophagosomes and LC3 accumulation spots, Atg5, LC3A, LC3B mRNA levels and LC3II/LC3I, Beclin-1 protein levels induced by Cd, and markedly elevated p62 mRNA and protein levels. Overall, the results verified that Cd induced [Ca2+ ]c overload mainly originated from ER Ca2+ release mediated by PLC-IP3 -IP3 R pathway, then triggered autophagy in duck renal tubular epithelial cells.

Decitabine attenuates ischemic stroke by reducing astrocytes proliferation in rats.

DNA methylation regulates epigenetic gene expression in ischemic stroke. Decitabine attenuates ischemic stroke by inhibiting DNA methylation. However, the underlying mechanism of this effect is not known. A model of ischemic stroke in Sprague-Dawley rats was induced through middle cerebral artery occlusion followed by reperfusion step. The rats were randomly treated with decitabine or vehicle by a one-time intraperitoneal injection. Sham rats received similar treatments. Four days after treatment, the rats were perfused with saline or 4% paraformaldehyde after which the brain was excised. DNA methylation level and brain infarct volume were determined by dot blot and histochemistry, respectively. The cellular co-localization and quantitative analysis of DNA methylation were assessed by immunohistochemistry and expression levels of cdkn1b (p27) mRNA and protein were measured by qRT-PCR and immunohistochemistry, respectively. The proliferation of astrocytes and number of neurons were determined by immunohistochemistry. Rats treated with decitabine showed hypomethylation and reduced infarct volume in the cortex. DNA methylation was decreased in astrocytes. Decitabine upregulated p27 mRNA and protein expression levels and attenuated the proliferation of astrocytes in vivo and vitro. Decitabine promotes p27 gene expression possibly by inhibiting its DNA methylation, thereby decreases the proliferation of astrocytes, neuronal death and infarct volume after ischemic stroke.

Hydroxy-γ-sanshool from Zanthoxylum bungeanum (prickly ash) induces apoptosis of human colorectal cancer cell by activating P53 and Caspase 8.

Sanshools, long-chain polyunsaturated amides in Zanthoxylum bungeanum (prickly ash), have important bioactivity. The objective was to assess inhibitory effects and molecular mechanisms of sanshools isolated from supercritical fluid (SCF) extract on human colon adenocarcinoma cells (HCT-116) cultured in vitro. Cells were exposed to various concentrations (0, 50, 90, or 130 μM) of sanshools for 24 or 48 h, with assessment of apoptosis and cell cycle arrest as well as regulatory gene and protein expression associated with apoptosis and the cell cycle. Sanshools profoundly inhibited growth of HCT-116 cells, with hydroxy-γ-sanshool (HRS) being the optimal active component (IC50 = 88.01 μM) inhibiting cell proliferation and having no cytotoxic effect to normal cells (IC50 = 481.52 μM) by CCK-8 assay. In HCT-116 cells, HRS inhibited cell growth, induced morphological distortion, and arrested the cell cycle at G1 phase (50.31 ± 4.13% vs. 72.16 ± 8.14% in Control and 130 μM HRS, respectively), and also caused programmed cell death in a dose-dependent manner. The percentage of apoptotic cells were remarkably increased after treated with HRS (6.2, 11.9, 19.8, and 30.7% for 0, 50, 90, and 130 μM, respectively). Moreover, in HCT-116 cells, HRS significantly inhibited mRNA and protein levels of Cyclin D1, CDK4, PCNA, and increased mRNA and protein levels of P21, P53, Fas, and Caspase 8. Furthermore, inhibitors of P53 and Caspase 8 proteins significantly mitigated the HRS-induced cell cycle arrest and apoptosis. In conclusion, our study provides evidence that HRS induced human colorectal cancer cell apoptosis by up-regulating P53 and Caspase 8.

Chronic restraint stress promotes gastric epithelial malignant transformation by activating the Akt/p53 signaling pathway via ADRB2.

The etiology of gastric cancer is associated with infectious, environmental and dietary factors, as well as genetic background. Additionally, emerging evidence has supported the vital role of chronic emotional stress on gastric carcinogenesis; however, the underlying mechanism remains unclear. The present study aimed to investigate the effects of chronic stress and a detrimental diet on gastric malignant epithelial transformation in rats. Therefore, 26 Wistar rats were randomly divided into the following four groups: i) Control; ii) detrimental diet (DD); iii) detrimental diet with chronic restraint (DR) and iv) detrimental diet with chronic restraint and propranolol treatment (DRP). ELISA was performed to detect the serum levels of epinephrine and norepinephrine. Epithelial cell apoptosis was analyzed using the TUNEL assay. The mRNA and protein expression levels of Akt and p53 were detected using reverse transcription quantitative PCR and western blotting, respectively. Pathological changes were analyzed using hematoxylin and eosin staining (H&E). The H&E staining results showed that dysplasia in the gastric mucosa occurred in two of eight rats in the DD group and in four of five rats in the DR group, whereas no dysplasia was detected in the DRP group. The apoptotic ratios of gastric epithelial cells were significantly decreased in all treatment groups compared with the control group. Adrenoceptor β2 (ADRB2) protein expression levels were increased significantly only in the DR group and this effect was significantly reduced in the DRP group. The mRNA expression levels of Akt and p53 were significantly upregulated in the DD group, and Akt mRNA expression was further elevated in the DR group. With regard to protein expression, the levels of Akt and p-Akt were significantly increased in the DR group, whereas these effects were reversed in the DRP group. Furthermore, the ratio of p-p53/p53 protein was significantly reduced in the DD or DR groups, but was reversed in the DRP group. Collectively, the findings of the present study suggested that chronic restraint stress potentially aggravates the gastric epithelial malignant transformation induced by a detrimental diet, at least partially via the Akt/p53 signaling pathway mediated via ADRB2.

Biochemical and Biological Evaluation of an L-Asparaginase from Isolated Escherichia coli MF-107 as an Anti-Tumor Enzyme on MCF7 Cell Line

Background: One of the most widely used anticancer agents is microbial L-ASNase. Herein, we assessed the biochemical and biological properties of an isolated L-ASNase from a Gram-negative bacteria strain, Escherichia coli MF-107. Methods:Using garden asparagus, we obtained several bacterial isolates. These strains were further screened for L-ASNase activity. A promising bacterial isolate was selected for L-ASNase production and subsequent purification. The molecular weight of purified L-ASNase was determined. The MTT assay was applied to assess the cytotoxic effect of the purified enzyme. Also, for caspase activity determination and the apoptotic effect of purified enzyme on in cells, we conducted a real-time PCR method. Results:The molecular weight of the enzyme was approximately 37 kDa. In the pH range of 7.5 to 8, the enzyme had considerable stability. At 35 °C, the purified L-ASNase optimum activity was recorded. The cytotoxic effect of the enzyme on treated cells was dose-dependent with an IC50 value of 5.7 IU/ml. The Bax gene expression considerably raised by 5.75-fold (p < 0.001) upon L-ASNase treatment. On the other hand, the anti-apoptotic Bcl-2 gene expression showed a 2.63-fold increase compared to the control (p < 0.05). It was detected that the mRNA levels of caspase-3 and p53 were considerably upregulated (5.93 and 1.85-fold, respectively). We did not find any alternation in the caspase-8 activity of the treated cells compared to untreated cells. Conclusion:In this research, the proliferation of the breast cancer cells remarkably inhibited via the cytotoxic effect of isolated L-ASNase from microbial sources.

Interleukin 7 inhibit autophagy via P53 regulated AMPK/mTOR signaling pathway in non-small cell lung cancer

Interleukin 7 (IL-7) has been demonstrated regulating lymphangiogenesis, apoptosis, and proliferation. Whether IL-7 induce or inhibit autophagy in non-small cell lung cancer (NSCLC) are unknown. In this study, Western blot was used to detect cytoplasmic and nuclear protein of p53, total protein of AMP-activated protein kinase (AMPK), mammalian target of rapamycin (mTOR) and Light chain 3 (LC3). Quantitative Real-Time PCR (qRT-PCR) was used to detect p53 mRNA level after treated with IL-7. Then using transmission electron microscopy to observe the morphological change of autophagosome. 123 cases of NSCLC were collected for survival analysis, immunohistochemistry staining and cox regression multivariate analysis. We find that IL-7 induce the p53 translocation from nucleus to cytoplasm, then IL-7 down-regulate phosphorylation of AMPK and up-regulate phosphorylation of mTOR. The expression of AMPK and p53 were associated with IL-7/IL-7R and mTOR expression. Clinically, AMPK and p53 were well correlated with stage and survival of lung cancer patients. IL-7R, mTOR and tumor stage were the strongest predictors of survival. In conclusion, IL-7 inhibit autophagy in NSCLC via P53 regulated AMPK/mTOR signaling pathway. AMPK and p53 are correlated with patients’ survival. IL-7R, mTOR and tumor stage are the strongest predictor of survival.

Piperine increases striatal levels of DA and TH and decreases α-syn and Aβ42 deposition in PDD mice by regulting autophagy: downexpression Beclin-1 and LC3B and upexpression p62

Piperine, the major pharmacological ingredient of pepper, can delay the procession of neuropharmacological effects, but its effects and mechanisms on Parkinson's disease dementia (PDD) mice is still unclear, we investigated whether piperine could help treating PDD mice. Here, PDD mice were randomly divided into eight groups (n = 12/group): a normal control group, a PDD model group, a madopar group, an autophagy inhibitor group, an autophagy activator group, and groups receiving low, medium or high doses of piperine respectively. The normal control and PDD model mice were injected with saline. Treatments were administered to the mice once per day continuously for 30 days. The behavioral tests were assessed. Dopamine (DA), Monoamine Oxidase-B (MAO-B), DOPA decarboxylase (DDC), β-secretase, acetylcholinesterase (AChE), amyloid β42 (Aβ42), tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) levels were detected. α-synuclein (α-syn), tyrosine hydroxylase (TH), HSP90, Beclin-1, LC3B, p62 mRNA levels and miRNA-99a-5p expression were determined. Neuronal histology was observed. The behavior of PDD mice improved significantly after peperine treatment compared with the PDD model mice. In addition, our results also showed that peperine treatment increased DA, TH, DDC and p62 levels, decreased MAOB, β-secretase, AChE, Aβ42, TNF-α, IL-6, Beclin-1 and LC3B levels, and down-regulated α-syn, HSP90, Beclin-1, LC3B mRNA levels and miR-99a-5p expression. These findings suggest that piperine may reduce the expression of mmu-miR-99a-5p and autophagy-related factors (HSP90, Beclin-1, LC3B and p62) to alleviate the neurological impairment of PDD mice, which is shown to slow down the process of DA metabolism and Aβ production and resist neuroinflammation.

Abstract 5373: Intact regulation of G1/S transition renders esophageal squamous cell carcinomas sensitive to PI3Kα inhibitors by blocking SKP2-mediated p21 degradation

Abstract Esophageal cancer is the seventh most common cancer and the sixth leading cause of cancer-related mortality in the world, of which about 80% are esophageal squamous cell carcinomas (ESCC). Mutation or amplification of PIK3CA frequently occurs in ESCC patients, indicating PI3Kα is a promising target for ESCC treatment. CYH33 is a novel PI3Kα-selective inhibitor, which is currently in clinical trials for the treatment of solid tumors including advanced ESCC. PI3Kα inhibitors including CYH33 and the marketed alpelisib displayed highly variable activity against the proliferation of a panel of ESCC cells, reflecting the heterogeneity of ESCC. We sought to discover predictive biomarkers for the efficacy of PI3Kα inhibitors in ESCC by evaluating its activity to against the growth of a panel of 16 clinically relevant patient-derived xenografts (PDXs). We found that PDXs with CCND1 amplification were more sensitive to CYH33, which was consistent with the positive correlation between CYH33 activity and CyclinD1 expression in PDXs detected by tissue microarray. As Cyclin D1 plays a critical role in G1/S transition in cell cycle progression, proteins regulating G1 phase were profiled in ESCC cells. Elevated expression of Cyclin D1, p21 and Rb was found in CYH33-sensitive cells compared to those in resistant cells, indicating intact G1/S regulation in sensitive cells. Accordingly, CYH33 significantly arrested sensitive cells at G1 phase, while it had little effect in resistant cells. The cellular level of p21 as well as p21 bound to the CDK4/6-Cyclin D1 or CDK2-Cyclin E complex increased upon CYH33 treatment in sensitive cells, which was accompanied with decreased phosphorylation of Rb. Though CYH33 had little effect on the mRNA level of p21, its treatment prolonged the half-life of p21 protein in sensitive cells. CYH33 was found to impede the interaction between the E3 ubiquitin ligase SKP2 and p21 in sensitive cells, resulting in decreased ubiquitination of p21. However, CYH33 failed to stabilize and accumulate p21 in resistant cells. In conclusion, we found that ESCC cells with intact G1 phase regulation were more sensitive to CYH33. CYH33 arrested sensitive ESCC cells at G1 phase via inhibiting the ubiquitination and degradation of p21 by SKP2, which further resulted in decreased activity of CDK4/6 and CDK2 as well as Rb phosphorylation. These findings revealed the insight mechanism of the differential activity CYH33 against ESCC and provided potential candidate biomarkers for the treatment of ESCC. Citation Format: Yuxiang Wang, Xu Zhang, Xi Zhang, Yanyan Shen, Qingyang Ma, Yuemei Qiao, Yi Wang, Biyu Yang, Lan Xu, Gaoxiang Ge, Landian Hu, Xiangyin Kong, Chunhao Yang, Jian Ding, Yi Chen, Linghua Meng. Intact regulation of G1/S transition renders esophageal squamous cell carcinomas sensitive to PI3Kα inhibitors by blocking SKP2-mediated p21 degradation [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 5373.