); [EMAIL_ADDRESS] (Z.O.) ###### Abstract  
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 to 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. Keywords: thymoquinone; UHRF1; ubiquitination; HAUSP; tumor suppressor genes +
Footnote †: journal: Applied Sciences  
1 Biochemistry Department, Faculty of Science, King Abdulaz University, Jeddah 21589, Saudi Arabia; [EMAIL_ADDRESS]. Chfr [14] and UHRF1, from autoubiquitination [15]. Indeed, HAUSP physically interacts with UHRF1 and an active HAUSP completely abolished the autoubiquitination of UHRF1 through the removal of ubiquitin adducts [15]. These findings indicate that HAUSP positively regulates the expression levels of UHRF1 through targeting the RING domain-mediated autoubiquitination activity of UHRF1, preventing proteasomal degradation of UHRF1 [15]. These data, taken together, suggest that an intact function of the UHRF1 RING domain is essential for cellular survival and its disruption through the dissociation of HAUSP from the UHRF1 complex significantly enhances the sensitivity of cancer cells to chemotherapeutic, making UHRF1 a promising target in the search for natural compound to target in cancer therapy. Several in vitro and in vivo studies have shown that UHRF1 downregulation in cancer is sufficient to induce apoptosis through the reactivation of various tumor suppressor genes (TSGs) [7; 16; 17; 18]. Indeed, UHRF1 expression decreases in response to several natural products, including napthiazarin [19], shikonin [20], curcumin [5], epigallocatechin-3-gallate (EGCG) [4], resveratrol [21], anisomycin [21], luteolin [22], doxorubicin [23] and thymquinone (TQ) [1; 11; 24; 25]. TQ, the most abundant biologically active component of black seed oil, shows inhibitory effects on many human cancer cells by targeting several signaling pathways, including the UHRF1/TSGs axis [1; 7; 11]. TQ was shown to induce the rapid autoubiquitination of UHRF1 in cancer cells through its RING domain, and this event was correlated with HAUSP downregulation [11], which is known to control the ubiquitination status of UHRF1 and protects it from degradation by the proteasome [15]. These findings suggest that HAUSP downregulation-mediated rapid autoubiquitination of UHRF1 in response to TQ is an essential first step for subsequent UHRF1 degradation [11].