No abstract

The tumor suppressor p73 is a member of p53 family and has a high degree of similarity with p53 function and structure. Like p53, p73 can also induce the expression of several genes involved in cell cycle and apoptosis. p73 expression is downregulated in many tumors by several mechanisms including the ubiquitination pathway. Thus, understanding the ubiquitin-proteasome pathway in p73 regulation will help in targeting this later and develop a new promising therapeutic strategy for cancer with p53 mutations. The aim of this study was to evaluate the effect of Thymoquinone (TQ), the major biologically active compound of the black seed oil on the expression of several E3 ubiquitin ligase enzymes known to be regulators of p73 and the related events in cancer cells with p53 mutation, such as the human acute lymphoblastic leukemia Jurkat cells, the human triple-negative breast cancer (MDA-MB-468 cells) and human promyelocytic leukemia HL60 cells. RNA-seq data showed that several E3 ubiquitin-ligase enzymes, well documented to be involved in the degradation of p73 including Itch, Pirh2, E3s Pin2, Mdm2, TRIM32 and SCFFBXO45 were downregulated in Jurkat cells. Among the target genes, Itch was significantly downregulated in TQ-treated Jurkat cells as compared with control cells. TQ-induced Itch downregulation was confirmed by real-time RT-PCR in Jurkat cells, MDA-MB-468 cells and HL60. Treating Jurkat cells with either TQ or the proteasome inhibitor MG132 induced an upregulation of p73. The present study indicates that TQ could be a promising inhibitor of the E3-ubiquitin ligase Itch leading to the upregulation of tumor suppressor p73 in cancers expressing mutant p53.

Downregulation of the ubiquitin-like containing PHD and ring finger 1 (UHRF1) oncogene in cancer cells in response to natural anticancer drugs, including thymoquinone (TQ), is a key event that induces apoptosis. TQ can induce UHRF1 autoubiquitination via the E3 ligase activity of its RING domain, most likely through the downregulation of herpes virus-associated ubiquitin-specific protease (HAUSP). In this study, we evaluated whether HAUSP downregulation and fast ubiquitination of UHRF1 are prerequisites for UHRF1 degradation in response to TQ in cancer cells and whether doxorubicin can mimic the effects of TQ on UHRF1 ubiquitination. RNA sequencing was performed to investigate differentially expressed genes in TQ-treated Jurkat cells. The protein expression of UHRF1, HAUSP and Bcl-2 was detected by means of Western blot analysis. The proliferation of human colon cancer (HCT-116) and Jurkat cells was analyzed via the WST-1 assay. RNA sequencing data revealed that TQ significantly decreased HAUSP expression. TQ triggered UHRF1 to undergo rapid ubiquitination as the first step in its degradation and the inhibition of its cell proliferation. TQ-induced UHRF1 ubiquitination is associated with HAUSP downregulation. Like TQ, doxorubicin induced a similar dose- and time-dependent downregulation of UHRF1 in cancer cells, but UHRF1 did not undergo ubiquitination as detected in response to TQ. Furthermore, TQ decreased Bcl-2 expression without triggering its ubiquitination. A fast UHRF1 ubiquitination is an indispensable event for its degradation in response to TQ but not for its responses to doxorubicin. TQ appears to trigger ubiquitination of UHRF1 but not of the Bcl-2 oncogene, thereby identifying UHRF1 as a specific target of TQ for cancer therapy.