Abstract. 5-Fluorouracil (5-FU) is the chemotherapeutic drug of choice for the treatment of metastatic colorectal cancer (CRC). Tumor suppressor candidate 4 (TUSC4), also referred to as nitrogen permease regulator-like 2 (NPRL2), is located at chromosome 3p21.3 and expressed in numerous normal tissues, including the heart, liver, skeletal muscle, kidney, and pancreas. The aim of the present study was to investigate the functional mechanism by which TUSC4 affects sensitivity to 5-FU and to determine its clinical significance in CRC. The results of the present study demonstrated that TUSC4 overexpression increases the sensitivity of HCT116 cells to 5-FU. The IC 50 of 5-FU was reduced in cells transduced with TUSC4 compared with negative control (NC) cells, and the effect of TUSC4 on 5-FU sensitivity was time dependent. Following TUSC4 transduction in HCT116 cells, a proportion of the cells were arrested in the G1 phase of the cell cycle, and a reduction in the S phase population was observed. Flow cytometry analysis revealed that TUSC4 transduction and 5-FU treatment increased apoptosis compared with NC cells. The mechanism through which TUSC4 overexpression enhances 5-FU sensitivity involves the downregulation of the function of the PI3K/Akt/mTOR network. Furthermore, 5-FU upregulated caspase-3 and caspase-9, promoting apoptosis in TUSC4-overexpressing cells compared with cells that were transduced with TUSC4 or treated with 5-FU and NC cells. The findings of the present study indicate that TUSC4 has potential as a biomarker for the prediction of the response to 5-FU and prognosis in patients with colorectal cancer and other types of human cancer. TUSC4 may also act as a molecular therapeutic agent for enhancing the patient's response to 5-FU treatment.

The nitrogen permease regulator-like-2 (NPRL2) gene is a candidate tumor suppressor gene, which has been identified in the 3p21.3 human chromosome region. Decreased expression levels of NPRL2 have been observed in colorectal cancer (CRC) tissues, however, the function of NPRL2 in CRC progression remains to be fully elucidated. The present study investigated the biological characteristics of the HCT116 and HT29 CRC cell lines overexpressing exogenous NPRL2. NPRL2 recombinant lentiviral vectors were also constructed and transfected in the present study. Cell growth was determined using a Cell Counting Kit-8 assay and a colony formation assay. The cell cycle and rate of apoptosis were assessed using flow cytometric analysis. Transwell assays were used to evaluate cell invasion. The protein expression of phosphorylated (p)-AKT and caspase 3, B-cell lymphoma 2 (Bcl2) and Bcl-2-associated X protein apoptosis-associated genes, were detected using western blotting. The results revealed that NPRL2 overexpression inhibited cell growth, induced cell cycle G1 phase arrest, promoted apoptosis and inhibited invasion in the two human CRC cell lines. Furthermore, the protein expression levels of p-AKT and Bcl2 were significantly reduced in the NPRL2-transfected HCT116 and HT29 cells, compared with the mock-transfected group and control group, while the protein expression of caspase-3 was increased. Therefore, NPRL2 acted as a functional tumor suppressor in the CRC cell lines.

Previous studies have suggested that hepatocyte apoptosis may be a fundamental underlying mechanism of liver injury and diseases, such as liver fibrosis. Relaxin‑3 has been reported to have anti‑fibrotic actions in the heart and to attenuate isoproterenol‑induced myocardial injury; however, the beneficial role of relaxin‑3 on hepatocyte apoptosis remains to be elucidated. The aim of the present study was to explore the role and possible mechanisms of relaxin‑3 through hydrogen peroxide (H2O2)‑induced apoptosis in primary human hepatocytes. Cells were treated with relaxin‑3 and then cell viability, morphological features, the presence of cleaved caspases as well as the levels of endoplasmic reticulum stress (ERS) protein markers and autophagy markers were evaluated. The H2O2 group showed significantly decreased cell viability, increased apoptosis as well as upregulation of caspases (cleaved caspase‑3, ‑8 and ‑9) and ERS protein markers compared with those of the control group. However, cells treated with relaxin‑3 (10 ng/ml) demonstrated improved cell viability, reduced apoptosis and decreased expression of cleaved caspases and ERS markers. However, the expression of autophagy markers remained unchanged following H2O2‑induced apoptosis and relaxin‑3 treatment. In conclusion, relaxin‑3 was shown to protect hepatocytes from H2O2‑induced apoptosis via downregulation of cleaved caspase‑8 and ‑9, as well as inhibition of the ERS pathway.

Colorectal cancer (CRC) is the third most common cancer worldwide. Chemotherapeutic compounds used for the treatment of CRC include oxaliplatin (L-OHP). While L-OHP improves CRC survival, certain patients are resistant. The nitrogen permease regulator like-2 (NPRL2) gene is a candidate tumor suppressor gene that resides in a 120-kb homozygous deletion region on chromosome 3p21.3. In the present study, it was demonstrated that NPRL2 overexpression increases the sensitivity of HCT116 cells to L-OHP. The IC50 of L-OHP was decreased in cells transduced with NPRL2 compared with negative control (NC) cells and the effect of NPRL2 on L-OHP sensitivity was time dependent. Following NPRL2 transduction in HCT116 cells, the cell cycle was arrested in the G1 phase and a partial decrease in the S phase population was observed. Flow cytometric analysis revealed that NPRL2 transduction and L-OHP treatment increased apoptosis compared with NC cells. The mechanism through which NPRL2 overexpression enhances L-OHP sensitivity involves downregulation of the functions of the phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin network. Furthermore, L-OHP upregulated caspase-3 and caspase-9 to promote apoptosis in NPRL2-overexpressing cells compared with cells that were transduced with NPRL2 or treated with L-OHP and NC cells (P<0.01). NPRL2 overexpression led to the downregulation of CD24, which could significantly reduce tumor invasiveness and decrease the metastatic capacity of HCT116 cells. These mechanisms are likely active in other types of cancer and may be exploited for the development of novel cancer therapies.

Abstract. The tumorigenesis and maintenance of a cancer cells is dependent upon the collaboration of multiple signaling pathways. Signal transducer and activator of transcription 3 (STAT3) and β-catenin are at the center of multiple cancer-associated signaling pathways; therefore, simultaneously targeting STAT3 and β-catenin may be a potential cancer treatment, leading to induced lethality of cancer cells. In the present study, HepG 2 liver cancer cells were transfected with small interfering RNA (siRNA) against β-catenin and STAT3 alone or in combination. The cell growth was assessed using an MTT assay and the levels of cell apoptosis were detected using flow cytometry. Protein levels of caspase-3, cleaved caspase-3, poly(ADP-ribose) polymerase (PARP) and cleaved PARP were determined using western blot analysis. Following siRNA transfection, β-catenin and STAT3 protein levels decreased at 72 h. HepG 2 cell growth inhibition and early apoptosis in the β-catenin and STAT3 siRNA co-transfection group were significantly greater than those in the groups transfected with β-catenin or STAT3 siRNA alone. Decreased caspase-3 and PARP levels, as well as enhanced cleavage of caspase-3 and PARP were observed in the β-catenin and STAT3 co-transfection group. Simultaneous silencing of β-catenin and STAT3 using siRNAs resulted in an enhanced loss of cell viability and induction of apoptosis in HepG 2 liver cancer cells, suggesting that these genes are promising targets for the further preclinical and clinical development of anti-cancer therapeutic strategies, which target several cancer signaling pathways simultaneously.