Delivery and Transcriptome Assessment of an In Vitro Three-Dimensional Proximal Tubule Model Established by Human Kidney 2 Cells in Clinical Gelatin Sponges.

The high prevalence of kidney diseases and the low identification rate of drug nephrotoxicity in preclinical studies reinforce the need for representative yet feasible renal models. Although in vitro cell-based models utilizing renal proximal tubules are widely used for kidney research, many proximal tubule cell (PTC) lines have been indicated to be less sensitive to nephrotoxins, mainly due to altered expression of transporters under a two-dimensional culture (2D) environment. Here, we selected HK-2 cells to establish a simplified three-dimensional (3D) model using gelatin sponges as scaffolds. In addition to cell viability and morphology, we conducted a comprehensive transcriptome comparison and correlation analysis of 2D and 3D cultured HK-2 cells to native human PTCs. Our 3D model displayed stable and long-term growth with a tubule-like morphology and demonstrated a more comparable gene expression profile to native human PTCs compared to the 2D model. Many missing or low expressions of major genes involved in PTC transport and metabolic processes were restored, which is crucial for successful nephrotoxicity prediction. Consequently, we established a cost-effective yet more representative model for in vivo PTC studies and presented a comprehensive transcriptome analysis for the systematic characterization of PTC lines.

Blockage of Nrf2 and autophagy by L-selenocystine induces selective death in Nrf2-addicted colorectal cancer cells through p62-Keap-1-Nrf2 axis.

Persistent Nrf2 activation is typically noted in many cancers, including colorectal cancer (CRC), aiding cancer cells in overcoming growth stress and promoting cancer progression. Sustained Nrf2 activation, which is beneficial for cancer cells, is called "Nrf2 addiction"; it is closely associated with malignancy and poor prognosis in patients with cancer. However, Nrf2 inhibitors may have adverse effects on normal cells. Here, we found that the selenocompound L-selenocystine (SeC) is selectively cytotoxic in the Nrf2-addicted CRC cell line WiDr cells, but not in non-Nrf2-addicted mesenchymal stem cells (MSCs) and normal human colon cells. Another CRC cell line, C2BBe1, which harbored lower levels of Nrf2 and its downstream proteins were less sensitive to SeC, compared with the WiDr cells. We further demonstrated that SeC inhibited Nrf2 and autophagy activation in the CRC cells. Antioxidant GSH pretreatment partially rescued the CRC cells from SeC-induced cytotoxicity and Nrf2 and autophagy pathway inhibition. By contrast, SeC activated Nrf2 and autophagy pathway in non-Nrf2-addicted MSCs. Transfecting WiDr cells with Nrf2-targeting siRNA decreased persistent Nrf2 activation and alleviated SeC cytotoxicity. In KEAP1-knockdown C2BBe1 cells, Nrf2 pathway activation increased SeC sensitivity and cytotoxicity. In conclusion, SeC selectively attacks cancer cells with constitutively activated Nrf2 by reducing Nrf2 and autophagy pathway protein expression through the P62-Nrf2-antioxidant response element axis and eventually trigger cell death.

Selenocystine induces oxidative-mediated DNA damage via impairing homologous recombination repair of DNA double-strand breaks in human hepatoma cells.

Selenocystine (SeC) has been identified as a novel compound with broad-spectrum anticancer activity. However, the effects of SeC on modifying DNA repair mechanism were less addressed. In this study, we demonstrated that SeC selectively induced cytotoxicity and genotoxicity against HepG2 hepatoma cell line. Comet assay revealed SeC-induced DNA damage in HepG2 cells, particularly in the form of DNA double strand breaks (DSBs), corroborated by the increase expression of the DSB marker, gamma-H2AX. We further demonstrated that SeC suppressed DNA homologous recombination repair, exacerbating DNA damage accumulation. Such effects on DNA damage and cell viability inhibition were alleviated by antioxidants, glutathione and Trolox, suggesting the involvement of reactive oxygen species (ROS). High levels of intracellular and mitochondrial ROS were detected in SeC-treated HepG2. In addition, SeC impaired the expression of antioxidant enzymes (superoxidase mutases and catalase), prompting the imbalance between antioxidant protection and excessive ROS formation and eliciting DSBs and cellular death. Decreased procaspase-3, 7, and 9 and Bcl-2 proteins and an increased Bax/Bcl-2 ratio, were observed after SeC treatment, but could be reversed by Torlox, confirming the action of SeC on ROS-induced apoptosis. In vivo, the xenograft tumor model of HepG2 cells validated the inhibition of SeC on tumor growth, and the induction of DSBs and apoptosis. In summary, SeC has the capability to induce ROS-dependent DNA damage and impeded DBS repair in HepG2 cells. Thus, SeC holds great promise as a therapeutic or adjuvant agent targeting DNA repair for cancer treatment.

4-Aminobiphenyl inhibits the DNA homologous recombination repair in human liver cells: The role of miR-630 in downregulating RAD18 and MCM8.

4-Aminobiphenyl (4-ABP), a well-known human carcinogen, has been shown to cause oxidative DNA damage and induce miR-630 expression in HepG2 cells treated with 18.75 µM-300 µM for 24 h. However, the underlying mechanism regarding the epigenetic regulation of miR-630 on DNA damage repair in liver cells is still not understood and needs to be investigated. In present study, our results showed that miR-630 was upregulated, resulting in mediating a decrease of DNA homologous recombination (HR) repair in L-02, HepG2 or Hep3B cells. Results from a luciferase reporting experiment showed that RAD18 and MCM8 were the potential targets of miR-630 during DNA damage induction. The downregulation of RAD18 or MCM8 by miR-630 was accompanied by inhibition of HR repair. Conversely, inhibiting miR-630 enhanced the expression of RAD18 and MCM8, and rescued HR repair. Additionally, we proved that the transcription factor CREB was related to miR-630 biogenesis in liver cells. Moreover, the levels of CREB, miR-630 expression, and double-strand breaks (DSBs) were attenuated by 5 mM N-acetyl-L-cysteine (NAC) pretreatment, indicating that reactive oxygen species (ROS)-dependent CREB-miR-630 was involved in DSB repair. These findings indicated that the ROS/CREB/-miR-630 axis plays a relevant role in the regulation of RAD18 and MCM8 in HR repair, which may facilitate our understanding of molecular mechanisms regarding the role of miR-630 downregulating DNA damage repair in liver cells.

Increase of lipid peroxidation by cisplatin in WI38 cells but not in SV40-transformed WI38 cells.

Cisplatin (CPT) is an effective anticancer drug that causes cumulative toxicity to normal tissues. It has been suggested that CPT damages normal cells by causing oxidative stress, but it is not known whether it can induce similar oxidative damage to tumor cells. In this study, by using normal human lung fibroblast (W138) cells and SV40-transformed WI38 (VA13) cells as a model, we compared the effect of CPT on cytotoxicity, apoptosis, lipid peroxidation, and mitochondrial gene expression, which could be regulated by oxidative stress, between normal and tumor cells. CPT induced greater growth inhibition and percentage of apoptotic cells in VA13 cells. However, levels of esterified F(2)-isoprostanes and 4-hydroxy-2-nonenal, two specific products of lipid peroxidation, were increased by CPT in WI38 cells, but not in VA13 cells. Furthermore, the transcript level of mitochondrial 12S rRNA was augmented by CPT in both cells, but to a higher degree in WI38 cells. The data suggest a correlation between lipid peroxidation and cytotoxicity or increased mitochondrial transcript levels in WI38 cells but not in VA13 cells. The results also indicate an altered response of oxidative damage and mitochondrial gene regulation to CPT in the transformed phenotype of WI38 cells.