Aberrant promoter CpG methylation and its translational applications in breast cancer.

Breast cancer is a complex disease driven by multiple factors including both genetic and epigenetic alterations. Recent studies revealed that abnormal gene expression induced by epigenetic changes, including aberrant promoter methylation and histone modification, plays a critical role in human breast carcinogenesis. Silencing of tumor suppressor genes (TSGs) by promoter CpG methylation facilitates cells growth and survival advantages and further results in tumor initiation and progression, thus directly contributing to breast tumorigenesis. Usually, aberrant promoter methylation of TSGs, which can be reversed by pharmacological reagents, occurs at the early stage of tumorigenesis and therefore may serve as a potential tumor marker for early diagnosis and therapeutic targeting of breast cancer. In this review, we summarize the epigenetic changes of multiple TSGs involved in breast pathogenesis and their potential clinical applications as tumor markers for early detection and treatment of breast cancer.

Prognostic significance of the Gustave Roussy immune (GRIm) score in cancer patients: a meta-analysis.

BACKGROUND: The prognostic value of the Gustave Roussy immune (GRIm) score in cancer patients has been widely reported but remains inconsistent. The aim of this study is to systematically investigate the relationship between the GRIm score and survival outcomes in cancer patients. METHODS: Relevant literature was identified using electronic databases including Web of Science, PubMed, and Embase from the inception to March 2023. The primary endpoints were long-term oncological outcomes. Subgroup analysis and sensitivity analysis were conducted during the meta-analysis. RESULTS: Fifteen studies (20 cohorts) including 4997 cancer patients were enrolled. The combined results revealed that patients in the high GRIm group had a deteriorated overall survival (HR = 2.07 95%CI: 1.73-2.48; p < 0.0001; I2 = 62%) and progression-free survival (HR = 1.42; 95%CI: 1.22-1.66; p < 0.0001; I2 = 36%). The prognostic values of GRIm on overall survival and progression-free survival were observed across various tumour types and tumour stages. Sensitivity analysis supported the stability and reliability of the above results. CONCLUSION: Our evidence suggested that the GRIm score could be a valuable prognostic marker in cancer patients, which can be used by clinicians to stratify patients and formulate individualized treatment plans.

The role of Crk/Dock180/Rac1 pathway in the malignant behavior of human ovarian cancer cell SKOV3.

Small GTPases, particularly the Rho family, are key regulators of cell motility and migration. Dock180 was well known for the main target of signal adaptor protein Crk and acted as a guanine-nucleotide exchange factor for small GTPase Rac1. In the present study, Dock180 was found to combine primarily with CrkI other than CrkII, and its association with Elmo1 was also demonstrated in ovarian cancer cell SKOV3. To evaluate the role of Dock180 in human ovarian cancer cell, we performed RNAi-mediated knockdown of Dock180 in SKOV3 cells using small interfering RNA expression vector. In Dock180 knockdown cells, we found that Elmo1 expression and Rac1 activity were decreased simultaneously. By contrast, the expressions of both another Crk-combining molecule C3G and Rap1 activity were observed to increase obviously. Accordingly, all Dock180 knockdown cells present with evident change in cell morphology, reduced cell proliferation, and attenuated cell migration. Taken together, these results suggest that signal transfer of Crk/Dock180/Rac1 is implicated in actin cytoskeleton reorganization and thus in the cell proliferation, motility, invasion, and of human ovarian cancer cell line SKOV3.

Attenuated Salmonella typhimurium carrying TRAIL and VP3 genes inhibits the growth of gastric cancer cells in vitro and in vivo.

BACKGROUND: Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL) and apoptin (VP3) of chicken anemia virus can selectively induce apoptosis in human tumor cell lines by two different pathways. Salmonella not only delivers functional genes to mammalian cells but also possesses antitumor activity and therefore could be adopted as a novel vector for anticancer therapy. MATERIALS AND METHODS: TRAIL and VP3 genes were cloned into a pBudCE4.1 vector and delivered by attenuated Salmonella typhimurium into gastric cancer cells, and their expression and antitumor effects in nude mice were monitored by Western blot, fluorescence microscopy, MTT assay, TUNEL staining, and immunohistochemistry. RESULTS: pBud-VP3 and pBud-TRAIL-VP3 plasmids were constructed to express TRAIL and apoptin in gastric cancer cells, leading to inhibition of cancer cell proliferation after 48 hours (P < 0.05). TRAIL and VP3 genes in pBudCE4.1 vector were also successfully delivered by attenuated S. typhimurium into gastric cancer cells in vivo, in which both TRAIL and apoptin were expressed. In vivo data indicated that S. typhimurium carring pBud-TRAIL-VP3 induced significant cell growth inhibition and tumor regression (P < 0.05). Moreover, expression of TRAIL and apoptin increased the expression of caspase-3 and caspase-9, resulting in enhanced apoptosis. CONCLUSION: Delivery of TRAIL and VP3 genes by attenuated S. typhimurium can significantly inhibit the growth of gastric cancer cells in vitro and in vivo.

[Overexpression of adiponectin prevents hepatocyte steatosis].

OBJECTIVE: To investigate the effect of adiponectin on hepatocyte steatosis. METHODS: L02 cells were transfected with pEGFP-N1-AdipoQ, a plasmid encoding pEGFP-adiponectin fusion protein, or pEGFP-N1. Lipid droplets in the hepatocytes were observed by oil red staining at 72 h. The contents of TG, FFA and glycerol in hepatocytes were measured. RESULTS: Compared to cells transfected with pEGFP-N1-AdipoQ plasmid, much more lipid droplets were observed in cells transfected with pEGFP-N1 plasmid. TG, FFA and glycerol contents in L02 cells and L02/pEGFP-N1 cells were significantly higher than those in L02/pEGFP-N1-AdipoQ cells. CONCLUSIONS: Overexpression of adiponectin prevent hepatocyte steatosis.

Long-term anti-inflammatory diet in relation to improved breast cancer prognosis: a prospective cohort study.

Inflammation-modulating nutrients and inflammatory markers are established cancer risk factors, however, evidence regarding the association between post-diagnosis diet-associated inflammation and breast cancer survival is relatively sparse. We aimed to examine the association between post-diagnosis dietary inflammatory index (DII®) and risks of all-cause and breast cancer-specific mortality. A total of 1064 female breast cancer survivors in the Prostate, Lung, Colorectal, and Ovarian Cancer Screening (PLCO) Trial prospective cohort, were included in this analysis if they had completed the diet history questionnaire (DHQ). Energy-adjusted DII (E-DIITM) scores were calculated based on food and supplement intake. Cox regression and competing risk models were used to estimate multivariable-adjusted hazards ratios (HRs) and 95% confidence intervals (95% CIs) by E-DII tertile (T) for all-cause and breast cancer-specific mortality. With median follow-up of 14.6 years, there were 296 (27.8%) deaths from all causes and 100 (9.4%) breast cancer-specific death. The E-DII was associated with all-cause mortality (HR T3 vs T1, 1.34; 95% CI, 1.01-1.81; P trend, 0.049, Table 2) and breast cancer mortality (HR T3 vs T1, 1.47; 95% CI, 0.89-2.43; P trend, 0.13; multivariable-adjusted HR for 1-unit increment: 1.10; 95% CI: 1.00-1.22). Non-linear positive dose-response associations with mortality from all causes were identified for E-DII scores (P non-linearity < 0.05). The post-diagnosis E-DII was statistically significantly associated with mortality risk among breast cancer survivors. Long-term anti-inflammatory diet might be a means of improving survival of breast cancer survivors.

[Anti-tumor effect of eukaryotic expressing plasmid containing soluble tumor necrotic factor-related apoptosis inducing ligand combined with human angiostatin Kringle (1 - 3) genes on human gastric cancer xenografts in nude mice].

OBJECTIVE: To investigate the anti-tumor effect of eukaryotic expressing plasmid containing human angiostatin Kringle (1 - 3) [hAG (K1-3)] combined with soluble tumor necrotic factor-related apoptosis inducing ligand (sTRAIL) genes on human gastric cancer xenografts in nude mice. METHODS: Recombinant plasmids of pBud-hAG and pBud-hAG-TRAIL were constructed by subcloning technique. Twenty nude BALB/c mice were inoculated with human gastric cancer cells of the line BGC-823 subcutaneously into the back. One week later after the appearance of implanted tumors the mice were randomly divided into 4 groups with pBud-hAG, pBud-hAG-TRAIL, pBud blank plasmid, and normal saline (NS) injected into the tumors respectively once the other day for 7 times. The size of tumor was observed. 7 days later the mice were killed with their tumors taken out. RT-PCR was used to detect the expression of hAG and sTRAIL. The microvessel density (MVD) of tumor was observed and recorded by detecting the protein of CD34 with immunohistochemistry on the microscopy. RESULTS: The MVD of tumor in the pBud-hAG-TRAIL and pBud-hAG groups were (4.8 +/- 0.9)/HP and (4.6 +/- 1.2)/HP respectively, significantly lower than those of the pBud and NS groups [(17.4 +/- 2.4)/HP and (18.2 +/- 2.7)/HP respectively, all P < 0.05], but there was no significant difference between the pBud-hAG-TRAIL and pBud-hAG groups. mRNA expression and protein expression of sTRAIL and hAG were positive in the pBud-hAG-TRAIL and pBud-hAG groups. The tumor volumes of tumors of the pBud-hAG-TRAIL and pBud-hAG groups were (1.325 +/- 0.012) cm(3) and (1.862 +/- 0.017) cm(3) respectively, both significantly lower than those of the pBud and NS groups [(3.637 +/- 0.032) cm(3) and (3.521 +/- 0.028) cm(3) respectively, all P < 0.05]. CONCLUSION: Angiostatin inhibits tumor angiogenesis through inhibiting the growth of vascular endothelial cells, and TRAIL induces tumor cell apoptosis. hAG (K1-3) combined with TRAIL can inhibit tumor growth more efficiently.

RNA interference-mediated silencing of the Hsp70 gene inhibits human gastric cancer cell growth and induces apoptosis in vitro and in vivo.

AIMS AND BACKGROUND: The role of heat shock protein (HSP) 70 in gastric cancer has been extensively examined in many studies for the past decade. It has been demonstrated that over-expression of Hsp70 might play important role in malignant transformation and maintenance of malignant phenotypes. Therefore, silencing the Hsp70 gene could be applicable in molecular therapies of human gastric cancer. Herein, we designed a small interfering RNA targeting Hsp70 to knock down its expression and investigated its effect on cell proliferation and apoptosis in a gastric cancer cell line. METHODS: Two plasmids (phsp1-siRNA, phsp2-siRNA), along with a negative control (phsp3-siRNA), were created using a genic recombination technique. BGC823 cell lines were used to perform experiments. Western blotting and RT-PCR were used to detect Hsp70 expression in vitro and in vivo. Cell morphology was observed under light microscope. Cell cycle and apoptosis were analyzed by flow cytometry and acridine orange/ethidium bromide double stain, and cell proliferative activity was measured by alamarblue assay. In all experiments, a negative control served as a baseline measure. RESULTS: We successfully constructed phsps-siRNA plasmids and transfected them into BGC823 cells. RT-PCR and western blotting revealed that the expression of Hsp70 was down-regulated in transfection groups compared with the control group. Flow cytometric analysis indicated that less S-phase fraction accumulated in small interfering RNA transfected cells than in parental cells and the cells transfected with empty vector. CONCLUSIONS: Our results demonstrated that RNAi against Hsp70 could effectively knock down gene expression, inhibit growth of cancer cells, induce cell cycle arrest and increase cell apoptosis in vitro and in vivo. Hsp70 might serve as a therapeutic target for human gastric cancer.

Mechanism of dihydroartemisinin-induced apoptosis in prostate cancer PC3 cells: An iTRAQ-based proteomic analysis.

AIMS: Prostate cancer (PCa) is one of the most common cancers in men in the world. Advanced PCa, especially castration-resistant PCa (CRPC), is difficult to cure. There is an urgent need to develop novel agents for CPRC. Dihydroartemisinin (DHA) is a semisynthetic derivative of artemisinin and is a well-known antimalarial drug. DHA has been documented to be a potential anticancer agent for PCa. However, the mechanisms underlying the anticancer activity of DHA are still unknown. MAIN METHODS: Proteomics analysis based on iTRAQ technology was performed to determine the protein profile changes in human prostate cancer PC3 cells treated by DHA, and apoptosis was detected by flow cytometry and transmission electron microscopy. KEY FINDINGS: DHA induced obvious apoptosis in PC3 cells. Using iTRAQ technology, we found 86 differentially expressed proteins linked to the cytotoxicity of DHA in PC3 cells. Gene ontology analysis showed the differentially expressed proteins were mainly associated with the protein synthesis and translation. Protein interaction network analysis and KEGG pathway analysis revealed altered aminoacyl-tRNA biosynthesis and metabolic pathways. Moreover, one candidate protein, heat shock protein HSP70 (HSPA1A), was identified by western blot analysis. SIGNIFICANCE: Our results indicate that multiple mechanisms involved in the anticancer activity of DHA in PC3 cells. Decreased HSP70 expression may have an important role in DHA-induced apoptosis in PC3 cells. Our data also provide novel insights into the anticancer mechanisms of DHA.

Aquaporin-9 downregulation prevents steatosis in oleic acid-induced non-alcoholic fatty liver disease cell models.

Aquaporin-9 (AQP9) is an aquaglyceroporin that acts as the adipose glycerol channel. However, the role of AQP9 in steatosis in non-alcoholic fatty liver disease (NAFLD) has not yet been fully elucidated. In the present study, the coding sequence of the AQP9 gene was obtained from LO2 cells by RT-PCR, and cloned into the pEGFP-N1 vector. Short hairpin RNA (shRNA) targeting the AQP9 gene was inserted into the pGenesil-1 vector. Recombinant plasmids were confirmed by enzyme digestion and sequence analysis, and transfected into cell models (derived from LO2 cells) of oleic acid-induced NAFLD. Our results demonstrated that AQP9 recombinant plasmids can be effectively expressed in cell models of NAFLD. Furthermore, in comparison with the control group, AQP9 overexpression significantly increased intracellular lipid content, triglyceride (TG), free fatty acid (FFA) and glycerol levels; however, the silencing of AQP9 exerted the opposite effects. Taken together, recombinant plasmids used to induce AQP9 overexpression and to silence AQP9 expression were successfully constructed. AQP9 overexpression aggravated the degree of steatosis; however, the silencing of AQP9 alleviated these effects. Based on these data, we suggest that AQP9 may serve as a novel molecular target for therapeutic intervention in NAFLD.

Epigenetic inactivation of PLCD1 in chronic myeloid leukemia.

Phospholipase C Î´1 (PLCD1), is located at the important tumor suppressor locus 3p22. It encodes an enzyme that mediates regulatory signaling of energy metabolism, calcium homeostasis and intracellular movements. PLCD1 has been studied in some human solid tumors relating to the CpG island methylation of the gene promoter as a functional tumor suppressor. However, no such information is available in chronic myeloid leukemia (CML). In this study, we investigated PLCD1 expression in the CML K562 cell line (0/1) and 15% (2/13) of bone marrow mononuclear cells with CML by using semi-quantitative PCR. The CpG island (CGI) methylation status of the PLCD1 promoter was detected in K562 (0/1) and 56% (23/41) of CML patients by methylation-specific PCR (MSP), but not in the normal adult bone marrow mononuclear cells. Furthermore, the DNA demethylation agent 5'-aza-2'deoxycytidine restored the expression of PLCD1 in K562 cells. Functional studies showed that ectopic expression of PLCD1 in K562 cells was able to dramatically inhibit their colony formation and induce cell cycle G1 arrest, suggesting that PLCD1 acts as a functional tumor suppressor and may serve as a biomarker for possible early detection and prognosis of CML.

Oral immunization with recombinant Mycobacterium smegmatis expressing the outer membrane protein 26-kilodalton antigen confers prophylactic protection against Helicobacter pylori infection.

Helicobacter pylori infection is prevalent worldwide and results in chronic gastritis, which may lead to gastric mucosa-associated lymphoid tissue lymphoma and gastric cancer. We have previously reported that oral immunization with recombinant Mycobacterium smegmatis expressing the H. pylori outer membrane protein 26-kilodalton (Omp26) antigen affords therapeutic protection against H. pylori infection in mice. In the present study, we investigated the prophylactic effects of this vaccine candidate on H. pylori challenge in mice. We found that oral immunization with recombinant Mycobacterium Omp26 significantly reduced H. pylori colonization in the stomach compared to inoculation with wild-type M. smegmatis in control mice. Six of the recombinant Mycobacterium-immunized mice (60%) were completely protected from H. pylori infection. The severity of H. pylori-associated chronic gastritis assessed histologically was significantly milder in mice vaccinated with recombinant Mycobacterium than in control animals. Mice immunized with recombinant Mycobacterium showed enhanced antigen-specific lymphocyte proliferation and antibody responses. Moreover, immunization with recombinant Mycobacterium resulted in an increased expression of interleukin-2 and gamma interferon in the stomach and spleen, as determined by reverse transcription-PCR analysis. Our results collectively suggest that vaccination with recombinant Mycobacterium Omp26 confers prophylactic protection against H. pylori infection. The inhibition of H. pylori colonization is associated with the induction of antigen-specific humoral and cell-mediated immune responses.

Recombinant Mycobacterium smegmatis mc(2)155 vaccine expressing outer membrane protein 26 kDa antigen affords therapeutic protection against Helicobacter pylori infection.

Orally administered recombinant Mycobacterium smegmatis (rM. smegmatis) vaccines represent an attractive option for mass vaccination programmes against various infectious diseases. Therefore, in the present study, we evaluated the capacity of the outer membrane protein 26kDa antigen (Omp26) of Helicobacter pylori (H. pylori) to induce therapeutic protection against H. pylori infection in mice. Omp26 was cloned and expressed in M. smegmatis mc(2)155 as a fusion with the Mycobacterium fortuitum beta-lactamase protein under the control of the up-regulated M. fortuitum beta-lactamase promoter, pBlaF. The rM. smegmatis strain was shown to be relatively stable in vitro in terms of plasmid stability and bacterial persistence. We found that oral immunization of H. pylori-infected mice with rM. smegmatis-Omp26 induced protection, i.e., significant reduction in bacterial colonization in the stomach. The protection was strongly related to serum specific antibodies with a Th(1) and Th(2) profile as well as to local cytokines in the stomach and spleen. These findings suggest that Omp26 is a promising vaccine candidate antigen for use in a therapeutic vaccine against H. pylori. The rM. smegmatis expressing Omp26 antigen could constitute an effective, low-cost combined vaccine against H. pylori.