Novel Insights into the Roles and Mechanisms of GLP-1 Receptor Agonists against Aging-Related Diseases.

Aging and aging-related diseases have emerged as increasingly severe health and social problems. Therefore, it is imperative to discover novel and effective therapeutics to delay the aging process and to manage aging-related diseases. Glucagon-like peptide-1 receptor agonists (GLP-1 RAs), one of the classes of antihyperglycemic drugs, have been recommended to manage type 2 diabetes mellitus (T2DM). Moreover, GLP-1 RAs have been shown to protect against oxidative stress, cellular senescence and chronic inflammation, which are widely accepted as the major risk factors of aging. However, their significance in aging or aging-related diseases has not been elucidated. Herein, we explain the underlying mechanisms and protective roles of GLP-1 RAs in aging from a molecular, cellular and phenotypic perspective. We provide novel insights into the broad prospect of GLP-1 RAs in preventing and treating aging-related diseases. Additionally, we highlight the gaps for further studies in clinical applications of GLP-1 RAs in aging-related diseases. This review forms a basis for further studies on the relationship between aging-related diseases and GLP-1 RAs.

[Research on promoter region methylation status of tumor repressor gene P16 in human hepatocellular carcinoma by using methylation sensitive restriction enzyme and semi-nested PCR assay].

OBJECTIVE: To construct a combination method of methylation sensitive restriction enzyme and semi-nested touch down PCR assay for studying the promoter region methylation status of P16 gene in human hepatocellular carcinoma. METHODS: According to the sequence of CpG rich promoter region of P16 gene, three primers were designed and synthesized for semi-nested touch down PCR assay to examine the promoter region methylation status of P16 gene. 340 bp segment of this region was cloned into vector pMD18-T; the plasmid was transformed into E. coli JM109 to harvest an extended quantity, then the plasmid was treated by CpG methylase M. Sss I, the methylated plasmid was named P16Pm+. This P16Pm+ is validated by digestion of Hpa II and is employed in studying the specificity and sensitivity of this constructed method. After construction, the method was used to examine the promoter region methylation status in P16 gene of 40 DNA samples from human HCCs and three DNA samples from normal human liver tissue. RESULTS: It was confirmed that the specificity and sensitivity of this method are solid and reliable (100 fg). It was found that 12/40 (30%) of hepatocellular carcinoma showed promoter region methylation in P16 gene whereas none (0/3) of the normal tissues was methylated in the promoter region in P16 gene. CONCLUSION: Promoter region methylation in P16 gene may take part in human hepatocellular carcinogenesis. The constructed method is simple, cost-effective and is of high specificity and sensitivity, thus suggesting its potential application to detecting promoter methylation in population-based studies.

[Growth inhibition and demethylation by a component of natural drug, CDP, in cancer cell lines].

OBJECTIVE: To investigate the inhibition of tumor cell lines' growth and the demethylation of p16 gene by a component of natural drug, CDP. METHODS: Human liver cancer cell line Huh-7 and breast cancer cell line T47D were treated with the CDP and DNA methyltransferase inhibitor, 5-azacytidine (5-aza-C). The inhibition of growth was assayed by MTT; the methylation status of p16 gene promoter was analyzed by MSP. RESULTS: 1. The CpG islands in promoter of p16 gene were identified as completely methylated in Huh-7 and T47D. 2. CDP and 5-aza-C inhibited the proliferation of Huh-7 and T47D cell lines in a dose, time-dependent mode. 3. Methylation-specific bands of CpG islands in p16 gene' promoter were still existed in Huh-7 and T47D, while unmethylation-specific bands appeared in Huh-7 after the cells being treated with 25, 50, 75 and 100 micromol/ L of CDP for 6 days as well as in T47D after the cells being treated with 50, 75 and 100 micromol/L of CDP for 6 days. 4. After the cells being treated with 50 micromol/L of CDP, the methylation-specific bands of CpG island in p16 gene' promoter were still existed in Huh-7 and T47D; unmethylation-specific bands appeared at 12 h and lasted to 144 h in Huh-7 while at 72 h and lasted to 144 h in T47D. CONCLUSION: CDP has inhibition effect on the cancer cell lines and has the ability to reverse the hypermethylated p16 gene promoter. These results suggest that CDP has great potential in the development of effective anticancer drugs.

[Detection of aberrant promoter CpG islands hypermethylation of GSTP1 in human primary hepatocellular carcinomas].

OBJECTIVE: To detect the aberrant promoter CpG islands hypermethylation status of GSTP1 gene in hepatocellular carcinomas (HCC) and to assess its significant role in hepatocarcinogenesis. METHODS: Surgically resected cancerous and non-cancerous liver tissues of 26 hepatocellular carcinoma patients were obtained from West China Hospital, and 11 peripheral blood samples from healthy donors as negative control were collected. Breast cancer cell line MCF-7 with CpG islands hypermethylation of GSTP1 as positive control was obtained from the Cell Bank of Chinese Academy of Sciences in Shanghai. All genomic DNA were extracted using common phenolchloroform approach, and the 5' CpG islands methylation status of GSTP1 gene was studied by methylation-specific polymerase chain reaction (MSP). RESULTS: GSTP1 gene promoter CpG island hyperthylation was detected in 88.5% (23/26) of cancerous tissues and in 69% (18/26) of corresponding non-cancerous tissues from the 26 HCC patients. None of the 11 control samples were methylation positive. CONCLUSION: The data indicates that the detection of GSTP1 gene methyl-lation may be a valuable biomarker by MSP for HCC early diagnosis and disease monitoring.

[Molecular mechanism of reversing metastatic phenotype in human high-metastatic large cell lung cancer cell line L9981 by nm23-H1].

BACKGROUND & OBJECTIVE: nm23-H1, a tumor metastasis suppressive gene, can reverse tumor metastasis phenotype. But the molecular mechanism of nm23-H1 in inhibiting or reversing metastasis of lung cancer is unclear. This study was to explore the molecular mechanism of nm23-H1 in reversing metastasis phenotype of lung cancer. METHODS: nm23-H1 gene and pLXSN were separately transfected into human lung cancer cell line L9981. Proliferation of L9981, L9981-pLXSN, and L9981-nm23-H1 cells was detected by MTT assay, cell invasive ability was detected by modified Boyden chamber. Tumorigenesis and experimental lung metastasis were determined in vivo. mRNA and protein levels of beta-catenin, E-Cadherin, CD44S, CD44V6, matrix metalloproteinase-2 (MMP-2), tissue inhibitor of metalloproteinase-1 (TIMP-1), and vascular endothelial growth factor (VEGF) were detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. RESULTS: (1)Cell proliferation, clone formation, and invasive ability were significantly lower in L9981-nm23-H1 cells than in L9981 cells [(19.5+/-2.9)% vs. 100%, 10.3+/-0.7 vs. 21.7+/-1.3, 31.0+/-3.0 vs. 151.0+/-6.3, P<0.01]. (2) The inhibitory rate of tumorigenesis of nude mice was significantly higher in L8891-nm23-H1 group than in L9981 group (85.6% vs. 0%, P<0.001)u the lung metastatic rate was significantly lower in L9981-nm23-H1 group than in L9981 group (0% vs. 100%, P<0.001). (3)nm23-H1 up-regulated mRNA and protein levels of beta-catenin, E-Cadherin, and TIMP-1, and down-regulated levels of MMP-2, CD44V6, and VEGF (P<0.01). (4) nm23-H1 up-regulated mRNA level of CD44s, protein level of CD44s didn't change (P>0.05). CONCLUSION: nm23-H1 gene can reverse malignant and metastatic phenotype of L9981 cells through regulating the expressions of lung cancer metastasis-related genes.

[Research on the growth inhibition of T47D cell and reversion of p16 hypermethylation by CDP].

OBJECTIVE: To inquire into the mechanism of CDP (a purified component of Chinese crude drug) in inhibiting the breast cancer T47D cell growth. METHODS: T47D cells were treated with different concentration of 5-azacytidine (5-aza-CR) and CDP for several days. The growth rate was assessed by cell proliferation experiment (MTT colorimetric assay). The changes in apoptic peak and cell cycle distribution were detected by flow cytometry (FCM). The levels of methylation and unmethylation status of p16 were detected by methylation specific PCR (MSP). RESULTS: After treatment with the two drugs, the cell growth rate decreased in a dose and time dependent manner (P<0.05). The cell cycle was influenced by the well-chosen concentration of 5-aza-CR (2 micromol/L) and CDP (50 micromol/L): the cell number increased from 65.1% to 71.3%, 84.3% in G0/G1 phase and decreased from 19.4% to 14.3%, 7.2% in S phase. Demethylation on p16 gene occurred after treatment with any of the two drugs for 6 days. CONCLUSION: CDP can reverse p16 hypermethylation and may hence inhibit the proliferation of tumor cells.

[The imprinting status and expression of insulin-like growth factor 2 gene in human hepatocellular carcinoma].

OBJECTIVE: To study the imprinting status and expression level of insulin-like growth factor 2 (IGF2) gene in hepatocellular carcinoma and to provide a clue for elucidating the mechanism of carcinogenesis of hepatocellular carcinoma. METHODS: The heterozygote status of IGF2 gene was detected by restriction fragment length polymorphism. The imprinting status and expression level of IGF2 were evaluated by semi-quantitative reverse transcription-polymerase chain reaction. RESULTS: It was found that 10/18 (55.6%) of hepatocellular carcinoma showed the gain of imprinting (GOI), with 6/10 (60%) adjacent cirrhosis of liver tissues also displaying GOI of IGF2. Overexpression of IGF2 in cancer tissues was detected in 9/18 (50%) samples, but no significant difference was observed among each imprinting status. CONCLUSION: GOI of IGF2 may take part in human hepatocellular carcinogenesis.

[Impact of p16INK4A and p15INK4B on human hepatocellular carcinoma cell proliferation and apoptosis].

OBJECTIVE: Both tumor suppressor p16INK4A and p15INK4B are members of INK family of CDK inhibitor. Although the role of p16 has been well documented, the role of p15 and its signaling pathway remain less well studied. This study was aimed to assess the effect of p16 and p15 on hepatocarcinoma cell lines with different status of Rb gene. METHODS: After identification of the genetic status of p16, p15 as well as Rb of human hepatocellular carcinoma (HCC) cell lines BEL7402, SMMC7721 with the use of multiple PCR, the eukaryotic expression p16 and p15 recombinants pXJ-p16 and pXJ-p15 were constructed, respectively. The existence of exogenous p16, p15 genes, and the expression of p16 and p15 were assayed by means of PCR and RNA dot blotting. Finally, the proliferation and apoptosis were studied by using MTT, colony formation assay and flow cytometry. RESULTS: Neither deletion of p16 nor p15 was detected in the two cell lines. However, Rb exons 14-16 instead of exons 22-23 deletion existed in SMMC7721. The increased mRNA expression level of p16 was found in BEL7402-p16 and SMMC7721-p16, while increased mRNA expression level of p15 was found in BEL7402-p15. The endogenous p16 and p15 genes were transcripted at low level. The cell growth and colony formation were decreased in BEL7402-p15, compared with either mock cell BEL7402 or vector control cell BEL7402-pXJ. Also shown in this study were an altered G1 phase population from 37.7% to 43.6%, an S phase population from 22% to 13% (P<0.05), and a Sub G1 peak (apoptosis peak) in BEL7402-p15. Conversely, BEL7402-p16 with endogenous p16 gene showed neither difference in cell cycle population nor difference in colony formation rate, compared with control cell groups. Additionally, SMMC7721-p16 cell growth was not inhibited by exogenous p16 gene. CONCLUSION: p15 significantly arrested cell proliferation and induced apoptosis in BEL7402 in vitro, and the function was not influenced by endogenous p15 gene. The inhibition of cell growth by p16 on HCC cells could be dependent on intact RB pathway.

Association of low p16INK4a and p15INK4b mRNAs expression with their CpG islands methylation with human hepatocellular carcinogenesis.

AIM: To study the significance of p16 and p15 transcription suppression with hypermethylation of their genes' 5'CpG islands during human hepatocellular carcinogenesis. METHODS: The mRNA expression levels of p16 and p15 genes were evaluated in cancerous, para-cancerous and non-cancerous tissues of 20 HCC, 3 normal liver tissues from 3 accidentally died healthy adults using semi-quantitatively Northern blot. The methylation status was also assessed with methylation specific PCR. RESULTS: p16 mRNA expression level was decreased in the cancerous tissues in 60% (12/20) of HCC patients, of which 2 cases had no p16 mRNA detected, 5 cases (25%) displayed variation in the order of cancerous

[Value of diagnosing micrometastasis by nested RT-PCR in the peripheral blood and bone marrow in non-small cell lung cancer patients].

OBJECTIVE: To evaluate the specificity, sensitivity and their clinical significance of detecting CK19 mRNA expression by nested RT-PCR for molecular diagnosis of micrometastasis in the peripheral blood and bone marrow of patients with non-small cell lung cancer. METHODS: CK19 mRNA expression was detected by nested RT-PCR in peripheral blood and bone marrow samples from 59 lung cancer patients, with samples of 11 benign pulmonary lesion patients and 20 healthy adults as control. RESULTS: The sensitivity of nested RT-PCR was 10(-6). The positive rates of micrometastasis were 33.89% (20/59) in peripheral blood and 22.03% (13/59) in bone marrow, with a highly positive correlation existing between the two groups (P < 0.05). The micrometastasis in peripheral blood and bone marrow was closely correlated with the pathological classification and cell differentiation (P < 0.05) and P-TNM stage (P < 0.01). No CK19 mRNA expression was found in the samples from patients with benign pulmonary lesion or healthy adult volunteers. CONCLUSION: The peripheral blood and bone marrow from patients with non-small cell lung cancer possesses micrometastasis that can not be detected by common methods. Nested RT-PCR technique shows favorable specificity and sensitivity in detecting the condition with definite clinical prospects.