Circulating-tumour DNA methylation of HAND1 gene: a promising biomarker in early detection of colorectal cancer.

BACKGROUND: Colorectal cancer (CRC) is one of the significant global health concerns with an increase in cases. Regular screening tests are crucial for early detection as it is often asymptomatic in the initial stages. Liquid biopsies, a non-invasive approach that examines biomarkers in biofluids, offer a promising future in diagnosing and screening cancer. Circulating-tumour DNA (ctDNA) is the genetic material in biofluids released into the circulatory system by cells. ctDNA is a promising marker for monitoring patients since cancer cells display distinct DNA methylation patterns compared to normal cells. The potential of our research to contribute to early detection and improved patient outcomes is significant. AIMS: The primary objective of this research project was to explore the HAND1 methylation levels in plasma ctDNA as a potential biomarker for diagnosing CRC and evaluate the methylation level of the well-established gene SPET9 to compare it with the methylation level of HAND1. MATERIALS AND METHODS: Plasma samples were collected from 30 CRC patients and 15 healthy individuals, with CRC samples obtained pre-treatment. ctDNA was extracted and treated with bisulfite for methylation status assessment. Quantitative methylation-specific PCR (qMS-PCR) was performed for HAND1 and SEPT9, using ß-actin (ACTB gene) as a reference. The study aims to evaluate the potential of these genes as diagnostic biomarkers for CRC, contributing to early detection and improved patient outcomes. RESULTS: Our study yielded significant results: 90% of CRC patients (27 out of 30) had hypermethylation in the SEPT9 gene, and 83% (25 out of 30) exhibited hypermethylation in the HAND1 gene. The methylation levels of both genes were significantly higher in CRC patients than in healthy donors. These findings underscore the potential of SEPT9 and HAND1 methylation as promising biomarkers for diagnosing CRC, potentially leading to early detection and improved patient outcomes. CONCLUSION: These findings highlight the potential of SEPT9 and HAND1 methylation as promising biomarkers for diagnosing CRC. However, further research and validation studies are needed to confirm these findings and to explore their clinical utility in CRC diagnosis and management.

Association of HOTAIR rs2366152 and rs1899663 polymorphisms with colorectal cancer susceptibility in Iranian population: A case-control study.

BACKGROUND: Despite the fact that numerous studies have investigated the association between genetic polymorphisms and colorectal cancer (CRC), more research is required to comprehend the molecular mechanisms of CRC. In the present study, we investigated the association between lncRNA HOTAIR rs2366152 and rs1899663 polymorphisms with CRC susceptibility in the Iranian population. METHODS: This case-control study consisting of 187 CRC patients and 200 healthy samples. The tetra-amplification refractory mutation system-polymerase chain reaction (Tetra-ARMS-PCR) technique was used for the genotyping of rs2366152 and rs1899663 polymorphisms. RESULTS: The findings showed that the AG genotype of the rs2366152 polymorphism has a protective effect on CRC susceptibility (OR = 0.60, 95% CI: 0.38-0.94, p-value = 0.023). Furthermore, rs2366152 polymorphism associated with CRC risk in an over dominant inheritance model (p-value = 0.0089). According to the outcomes of the rs1899663 polymorphism, the GT genotype had protective effects on CRC risk (OR = 0.55, 95% CI: 0.35-0.86, p-value = 0.008). Moreover, statistical analysis has shown that the rs1899663 polymorphism was associated with CRC risk in dominant (p-value = 0.013) and overdominant (p-value = 0.0086) inheritance models in the Iranian population. CONCLUSION: This study confirmed that HOTAIR rs2366152 and rs1899663 polymorphisms associated with CRC risk in different inheritance models. It is indeed necessary to do additional research to verify our findings.

Lapatinib Decreases the Preimplantation Aneuploidy Rate of in vitro Fertilized Mouse Embryos without Affecting Completion of Preimplantation Development.

One of the major reasons for implantation failure and spontaneous abortion is a high incidence of preimplantation chromosomal aneuploidy. Lapatinib simultaneously inhibits EGFR and HER2, leading to apoptosis. We hypothesized a higher sensitivity for aneuploid cells in preimplantation embryos to lapatinib based on reports of aneuploid cell lines being sensitive to some anticancer drugs. Late 2-cell mouse embryos were treated with lapatinib after determining a nontoxic dose. Morphologies were recorded 24, 48, and 60 hours later. The effect of lapatinib on the aneuploidy rate was evaluated by studying blastocyst cells using FISH. Although the rate of development to 8-cell and morula stage was higher in the control group (p < 0.05), there was no difference in development to the blastocyst stage at the same studied intervals between lapatinib-treated and control groups (p = 0.924). The mean number of cells in morula and blastocyst stages were not different between the groups (p = 0.331 and p = 0.175, respectively). The frequency of aneuploid cells and diploid embryos was, respectively, significantly lower and higher in lapatinib-treated embryos, (p < 0.001). Since lapatinib treatment reduced the aneuploidy rate without impact on the development of mouse preimplantation embryos to the blastocyst stage and number of total cells, lapatinib seems useful for prevention of preimplantation aneuploidy in in vitro fertilization.

Verbascoside Attenuates Rac-1 and HIF-1α Signaling Cascade in Colorectal Cancer Cells.

OBJECTIVE: Metastasis phenotype is considered as the main challenge in colon cancer therapeutic methods. Furthermore, the side effects of conventional colorectal cancer treatment methods have attracted a lot of attention into natural ingredients. The aim of the study was to assess the molecular mechanism of verbascoside as natural bio-compound in human HT29 colon cancer cells. METHODS: HT29 cells were cultured in RPMI-1640 medium containing 10% FBS and 1% penicillin/ streptomycin at 37°C and 5% CO2. HT-29 cells were treated with different concentrations of verbascoside (10, 20, 30, 40, 50, 70, 100 µg/ml) for 24 hours, then MTT assay was used to calculate 50% inhibitory concentration. The migration of the colon cancer cells was evaluated by scratch assay. To evaluate involved antiproliferative mechanism, Rac-1 (Ras-related C3 botulinum toxin substrate 1) and HIF-1α (hypoxia-inducible factor-1α) related gene expression were evaluated by Real Time PCR. RESULTS: The results showed that verbascoside inhibited HT29 colon cancer cell proliferation dose-dependently and IC50 was evaluated as 50 µg/ml (***P<0.001). The results of wound healing assay demonstrated verbascoside decreased cell migration in a dose dependent manner. In the IC50 treated HT29 cells metastatic progression was significantly suppressed as **P<0.01. The results of Real Time PCR showed an attenuating effect of verbascoside on Rac-1, Zeb-1 (zinc finger E-box binding homeobox 1), Arp2 (Actin-Related Proteins), Pak1 (p21 (RAC1) activated kinase 1), VEGF (Vascular endothelial growth factor) and HIF-1α as Epithelial-Mesenchymal Transition markers. The down regulation of mRNA levels was Rac-1= 15.38, HIF-1 α = 16.66, Pak-1, Arp-2= 6.25, VEGF=24.39, Zeb-1=35.71 in HT29 cells treated with IC50 concentration of verbascoside. CONCLUSION: Colorectal cancer cells induce Rac-1 and HIF-1α overexpression which plays an important role in the activation and progression of cell motility, angiogenesis and metastasis. Overall results showed that verbascoside elucidated significant anti-metastatic and anti-invasion activities through suppression of Rac-1, HIF-1α, and Zeb-1 signaling pathway and it may be a suitable candidate to overwhelm colon cancer metastatic phenotype.