Sarala Manandhar

Acquisition of doxorubicin resistance in ovarian carcinoma cells accompanies activation of the NRF2 pathway.

It has been firmly established that the transcription factor NRF2 is a critical element in the survival of healthy cells in response to oxidative stress because it up-regulates a wide array of antioxidant genes by binding to the antioxidant-response element (ARE). However, adaptive activation of the NRF2 system after an exposure of cancer cells to chemotherapy can be hypothesized, implying the acquisition of chemoresistance by tumors. In this study we have investigated the potential role of NRF2 signaling in the development of acquired resistance to doxorubicin. The human ovarian carcinoma cell line A2780, which is highly sensitive to doxorubicin, showed low levels of ARE binding and ARE-driven luciferase activity, as well as repressed expression of its target genes compared with resistant ovarian carcinoma SKOV3 and OV90 cells. Doxorubicin-resistant A2780DR cells, established after exposure to stepwise increasing concentrations of doxorubicin, displayed a refractoriness to doxorubicin-induced cell death. Acquisition of doxorubicin resistance in A2780 cells was accompanied by an elevation in NRF2 activity and consequent increase in the expression of the catalytic subunit of gamma-glutamylcysteine ligase and total GSH content. A critical role for NRF2 in the acquired chemoresistance of A2780DR cells could be confirmed by the restoration of doxorubicin sensitivity after stable expression of NRF2-specific shRNA in A2780DR cells, whereas inhibition of NRF2 could not further enhance doxorubicin sensitivity in the parental A2780 cells. These results suggest that the level of NRF2 activity might be a determining factor for doxorubicin sensitivity in ovarian carcinoma cell lines and adaptive activation of the NRF2 system can participate in the development of acquired resistance to anthracycline therapy.

NRF2 inhibition represses ErbB2 signaling in ovarian carcinoma cells: Implications for tumor growth retardation and docetaxel sensitivity

NF-E2-related factor 2 (NRF2) is a transcription factor that regulates the expression of various antioxidant and detoxifying enzymes. Although the benefit of NRF2 in cancer prevention is well established, its role in cancer pathobiology was recently discovered. In this study, the role of NRF2 in tumor growth and docetaxel sensitivity was investigated in ErbB2-overexpressing ovarian carcinoma SKOV3 cells. Interfering RNA-mediated stable inhibition of NRF2 in SKOV3 cells repressed NRF2 signaling, resulting in cell growth arrest at G(0)/G(1) phase and tumor growth retardation in mouse xenografts. Microarray analysis revealed that ErbB2 expression is substantially reduced in NRF2-inhibited SKOV3 and this was further confirmed by RT-PCR and immunoblot analysis. Repression of ErbB2 led to a decrease in phospho-AKT and enhanced p27 protein, reinforcing the effect of NRF2 knockdown on SKOV3 growth. Furthermore, NRF2 inhibition-mediated ErbB2 repression increases the sensitivity of these cells to docetaxel cytotoxicity and apoptosis. The linkage between NRF2 and ErbB2 was confirmed in the ErbB2-positive breast cancer cell line BT-474: NRF2 knockdown suppressed ErbB2 expression and enhanced docetaxel sensitivity. Our results provide insight into the coordinated regulation of signaling molecules responding to environmental stress and suggest that NRF2 modulation might be a therapeutic strategy to limit tumor growth and enhance sensitivity to taxane-based chemotherapy.

Effect of stable inhibition of NRF2 on doxorubicin sensitivity in human ovarian carcinoma OV90 cells

The transcription factor NRF2 defends the cell from oxidative stress by up-regulating a large number of antioxidant genes through its binding with antioxidant response element on gene promoters. Cancer cells are known to possess high levels of antioxidant genes that increases survival in cancer microenvironment of oxidative stress, particularly in the treatment with anticancer agents. In the current study we have examined the role of the NRF2 in doxorubicin sensitivity and tumor growth by establishing stable cell line expressing NRF2 shRNA in the human ovarian carcinoma cell line OV90. On knockdown of NRF2 through NRF2-specific shNRF2 expressing lentiviral plasmid, antioxidant response element-driven luciferase activity as well as the expression of NRF2-target genes were significantly suppressed compared to nonspecific scrambled RNA (scRNA) expressing cells. In addition, shNRF2 expressing OV90-shNRF2 cells showed a reduction in total GSH levels by 82% and cell growth was observed to be significantly retarded compared to scRNA control cells. Furthermore, stable inhibition of NRF2 sensitized OV90 cells were seen following doxorubicin treatment as shown by the analysis with MTT assay and propidium iodide-fluorescence-activated cell sorting. OV90-shNRF2 cells showed higher levels of cell death and apoptosis in response to doxorubicin than OV90-scRNA cells. While, when BALBc (nu/nu) mice with OV90 tumor xenograft in the flanks were injected with NRF2 shRNA containing viral particles and treated with doxorubicin a pattern of retardation in tumor growth was seen in shRNA group compared to scRNA group, but this difference was not statistically significant. In conclusion, we propose that the NRF2 signaling might be a molecular target to repress tumor growth and enhance cytotoxic effects of anticancer agent in cancer cells.

Activation of the Nrf2-antioxidant system by a novel cyclooxygenase-2 inhibitor furan-2-yl-3-pyridin-2-yl-propenone: implication in anti-inflammatory function by Nrf2 activator.

Furan‐2‐yl‐3‐pyridin‐2‐yl‐propenone (FPP‐3) is a novel synthetic compound and has demonstrated anti‐inflammatory activity by inhibiting cyclooxygenase‐2 (COX‐2). It is widely accepted that reactive oxygen species (ROS) generated by activated inflammatory cells can exacerbate inflammation. In this study, the potential antioxidative efficacy of FPP‐3 has been investigated in murine cells. FPP‐3 increased the expression of multiple antioxidative enzymes, including NAD(P)H:quinone oxidoreductase 1 (Nqo1), γ‐glutamylcysteine ligase (GCL) and heme oxygenase‐1 (HO‐1), by facilitating the nuclear translocation of nuclear factor‐erythroid 2‐p45‐related factor 2 (Nrf2). Inducibility of antioxidant proteins such as HO‐1 were lost in nrf2‐deficient murine fibroblasts. As a result of enhanced cellular antioxidative capacity, elevation of NF‐κB‐driven reporter gene expression by lipopolysaccharide was attenuated by FPP‐3 treatment in murine fibroblasts. Furthermore, FPP‐3 treatment inhibited UVA‐mediated induction of COX‐2 in murine keratinocytes. Our current study suggests that FPP‐3, which has been developed as a novel COX‐2 inhibitor, has antioxidative properties by activating the Nrf2‐ARE pathway. The dual function of this compound may provide a better strategy to block/attenuate the inflammation process and to alleviate ROS‐associated inflammatory complications.

Abstract 1661: Knockdown of Nrf2 in cancer cells suppresses tumor growth: implication for the inhibitory role in HIF-1α and ErbB2 signaling

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Nuclear factor-erythroid 2 (Nrf2) is the transcription factor regulating multiple antioxidant proteins and phase 2 detoxifying enzymes, and plays a crucial role in the cytoprotection against oxidative stress-associated damages. However, a potential role of Nrf2 in cancer biology has been emerged recently. In our mouse xenografts study, a stable knockdown of Nrf2 in cancer cells from the colon (HCT116 and HT29) and ovary (SKOV-3) strongly suppressed tumor growth. As an underlying mechanism, it was observed that a vessel formation and vascular endothelial growth factor (VEGF) expression were suppressed in Nrf2-inhibited colon cancer tumors. Suppressive effect of Nrf2 knockdown on angiogenesis was further evidenced by the chicken embryo chorioallantoic membrane assay and endothelial tube formation assay. The subsequent mechanism studies revealed that Nrf2-inhibited HT29 cells failed to accumulate HIF-1α protein under 1% O2 hypoxia, resulting in the limitation of the expression of HIF-1α target genes for angiogenesis. Whereas, a tumor growth suppression shown in SKOV-2 cells was associated with repressed expression of ErbB2 and the consequent increase in p27. Further experimental demonstrations showed that the expression of ErbB2 is inversely related with Nrf2 in other types of cancer cell lines; therefore, the cell growth is attenuated in these Nrf2-inhibited cancer cells. Taken together, our current study suggests that a stable Nrf2 inhibition in cancer cells suppresses tumor growth through the alterations in HIF-1α and ErbB2 signaling. Hence, Nrf2 may be a promising target to control cancer cell growth and chemoresistance. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1661. doi:10.1158/1538-7445.AM2011-1661