188Rhenium Treatment Induces DACT2 Expression in Hepatocellular Carcinoma Cells.

Objective: Epigenetic alterations, including any change in DNA methylation pattern, could be the missing link of understanding radiation-induced genomic instability. Dapper, Dishevelled-associated antagonist of ß-catenin homolog 2 (DACT2) is a tumor suppressor gene regulating Wnt/ß-catenin. In hepatocellular carcinoma (HCC), DACT2 is hypermethylated, while methylation status of its promoter regulates the corresponding expression. Radionuclides have been used to reduce proliferation and induce apoptosis in cancerous cells. Epigenetic impact of radionuclides as therapeutic agents for treatment of HCC is still unknown. The aim of this study was to evaluate epigenetic impact of 188Rhenium perrhenate (188ReO4) on HCC cells. Materials and Methods: In this in vitro experimental study, HepG2 and Huh7 cells were treated with 188ReO4, receiving 55 and 73 Mega Becquerel (MBq) exposures, respectively. For cell viability measurement, live/dead staining was carried out 18, 24, and 48 hours post-exposure. mRNA expression level of ß-Catenin, Wnt1, DNMT1, DACT2 and WIF- 1 genes were quantified by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Then, possible regulatory impact of DACT2 upregulation was investigated through evaluating methylation-specific PCR (MS-PCR). Results: Results showed that viability of both cells was reduced after treatment with 188ReO4 at three time points postexposure compared to the control groups. The qRT-PCR results showed that DACT2 mRNA level was significantly increased at 24, and 48 hours post-exposure in HepG2 cells compared to the control group, while, no significant change was observed in Huh7 cells. Methylation pattern of DACT2 promoter remained unchanged in HepG2 and Huh7 cells. Conclusion: Treatment with 188ReO4 reduced viability of HepG2 and Huh7 cells. Although DACT2 expression was increased after 188ReO4 exposure in HepG2 cells, methylation pattern of its promoter remained unchanged. This study assessed impacts of the 188ReO4 ß-irradiation on expression and induction of DACT2 epigenetic aberrations as well as the correlation of this agent with viability of cells.

Rhenium Perrhenate (188ReO4) Induced Apoptosis and Reduced Cancerous Phenotype in Liver Cancer Cells.

Recurrence in hepatocellular carcinoma (HCC) after conventional treatments is a crucial challenge. Despite the promising progress in advanced targeted therapies, HCC is the fourth leading cause of cancer death worldwide. Radionuclide therapy can potentially be a practical targeted approach to address this concern. Rhenium-188 (188Re) is a ß-emitting radionuclide used in the clinic to induce apoptosis and inhibit cell proliferation. Although adherent cell cultures are efficient and reliable, appropriate cell-cell and cell-extracellular matrix (ECM) contact is still lacking. Thus, we herein aimed to assess 188Re as a potential therapeutic component for HCC in 2D and 3D models. The death rate in treated Huh7 and HepG2 lines was significantly higher than in untreated control groups using viability assay. After treatment with 188ReO4, Annexin/PI data indicated considerable apoptosis induction in HepG2 cells after 48 h but not Huh7 cells. Quantitative RT-PCR and western blotting data also showed increased apoptosis in response to 188ReO4 treatment. In Huh7 cells, exposure to an effective dose of 188ReO4 led to cell cycle arrest in the G2 phase. Moreover, colony formation assay confirmed post-exposure growth suppression in Huh7 and HepG2 cells. Then, the immunostaining displayed proliferation inhibition in the 188ReO4-treated cells on 3D scaffolds of liver ECM. The PI3-AKT signaling pathway was activated in 3D culture but not in 2D culture. In nude mice, Huh7 cells treated with an effective dose of 188ReO4 lost their tumor formation ability compared to the control group. These findings suggest that 188ReO4 can be a potential new therapeutic agent against HCC through induction of apoptosis and cell cycle arrest and inhibition of tumor formation. This approach can be effectively combined with antibodies and peptides for more selective and personalized therapy.

Genome instability in AZFc region on Y chromosome in leukocytes of fertile and infertile individuals following exposure to gamma radiation.

PURPOSE: Men are exposed to various doses of ionizing radiation due to living in regions with high natural background radiation, accidentally, occupationally or for cancer treatment. To study genomic instability of AZFc region to gamma radiation, blood samples from normal, oligozoospermia, and azoospermia individuals were irradiated by a Co-60 source. METHODS: Irradiated cells were kept for 48 h in order to repair initial DNA damages. Real time PCR was performed for three markers (SY 1206, SY 1197, SY 579) for testing copy number variation before and after irradiation. Copy number variations were compared by calculation of cycle threshold comparative method. RESULTS: Copy number variations of studied markers in AZFc region (microdeletion and duplication) in all samples after exposure to radiation increased with a dose dependent fashion. The frequency of instability was significantly higher in samples from infertile men in comparison with fertile ones (p < 0.001). CONCLUSION: No significant difference was seen between the two infertile groups (p > 0.05). This observation might be a possible explanation for induction of azoospermia and oligozoospermia after radiotherapy. Increased frequency of induced microdeletion and duplication in infertile men compared with normal might be attributed to the deficiency in repair systems and the genetic factors involved in incomplete spermatogenesis of infertile men.

Increased frequency of micronuclei in lymphocytes of infertile males after exposure to gamma irradiation: a possible sign of genomic instability.

PURPOSE: Induced chromosomal instability and micronucleus (MN) formation in blood lymphocytes of infertile men in comparison with fertile men exposed to gamma radiation was investigated. METHODS: Blood samples of healthy and infertile donors were irradiated by 2 and 4 Gy Co-60 gamma-rays, then cultured in RPMI-1640 complete medium containing 1% phytoheamaglutinin (PHA) and incubated in a CO(2) incubator. Cytochalasin-B was added to the cultures at a final concentration of 4 µg/ml. Finally, harvesting, slide making, and analysis were performed according to standard procedures. RESULTS: We observed a statistically significant difference between the frequencies of micronuclei in lymphocytes of infertile individuals, compared to healthy donors, before and after exposure to gamma rays. Although higher in azoospermia patients, the frequency of MN was not statistically different between infertile groups. CONCLUSIONS: This study indicates that genomic instability in infertile men could probably contribute to the development of an impaired reproductive capacity.