## 3 Results
### Doxorubicin Induced Ubiquitination Independently of UHRF1 Downregulation in Cancer Cells  
The anticancer drug doxorubicin is commonly considered to exert its anti-tumor activity through targeting several signaling pathways, including the induction of the polyubiquitination of various proteins [32; 33; 34]. Doxorubicin was shown to decrease the expression of the UHRF1 protein in HCT-116 cells through an unknown mechanism [23]. Thus, we wanted to know whether doxorubicin induces a rapid ubiquitination of UHRF1 in cancer cells, thereby mimicking the effects of TQ on the ubiquitination status of UHRF1. As shown in Figure 3A, treatment of Jurkat cells with doxorubicin resulted in a dose-dependent decrease in the expression protein level of UHRF1 starting at doxorubicin concentrations of 0.25 \(\mu\)g/mL. A kinetic analysis of UHRF1 expression in Jurkat cells was then conducted using doxorubicin at 2 \(\mu\)g/mL, a concentration at which UHRF1 became undetectable after 24 h of treatment (Figure 3A). A time-course analysis showed that doxorubicin induced a sharp decrease in the expression of UHRF1 at 6 h (Figure 3B). However, although TQ at 10 min induced UHRF1 ubiquitination, as reflected by the rapid appearance of high-molecular-weight bands of UHRF1 (Figure 3C), Western blot analysis revealed that doxorubicin treatment did not challenge UHRF1 to perform ubiquitination before the onset of its degradation in Jurkat cells (Figure 3B). The same findings were observed in HCT-116 cells treated with doxorubicin (Figure 4). Figure 4A shows that the treatment of colon cancer cells with doxorubicin resulted in a dose-dependent decrease in the protein expression level of UHRF1, with concentrations as low as 10 \(\mu\)g/mL. The time-course effects of 60 \(\mu\)g/mL of doxorubicin on UHRF1 expression showed a decrease in the expression of UHRF1 starting at 6 h (Figure 4B). Again, the doxorubicin-induced UHRF1 downregulation was not correlated with the ubiquitination process, as reflected by the lack of high-molecular-weight bands of UHRF1 (Figure 4B), as was observed in response to TQ (Figure 4C). Figure 3: Dose- and time-dependent effects of doxorubicin and TQ on the protein expression of UHRF1 and ubiquitinated UHRF1 in Jurkat cells. (**A**) Jurkat cells were exposed to increasing concentrations of doxorubicin for 24 h. (**B**) Cells were exposed to 2 \(\mu\)g/mL doxorubicin for different times. (**C**) Cells were exposed to 30 \(\mu\)M TQ for 10 min. Expression of UHRF1 was analyzed via Western blotting, as described in the Materials and Methods. Taken together, these finding suggest that TQ and doxorubicin decreased UHRF1 protein expression through different mechanisms and indicate that fast UHRF1 ubiquitin is a prerequisite for its degradation in cancer cells in response to TQ but not for the response to doxorubicin. TQ Induced the Downregulation of the Bcl-2 Oncogene in Cancer Cells through a Ubiquitin-Independent Mechanism  
The B-cell lymphoma 2 (Bcl-2) oncogene is overexpressed in several tumors, including colon cancer [35; 36; 37; 38] and Jurkat cells [39; 40; 41]. Bcl-2 was shown to be degraded in the proteasome through a ubiquitination pathway in response to several signals [42; 43]. We investigated whether Bcl-2, like UHRF1, also underwent fast ubiquitination before its degradation in response to TQ in colon cancer cells and Jurkat cells. Dosage analysis of TQ on the expression of Bcl-2 in both cancer cell lines revealed that the treatment of HCT-116 cells (Figure 5A) and Jurkat cells (Figure 5B) with TQ induced a dose-dependent decrease in the protein level expression of Bcl-2, starting at 300 uM TQ in HCT-116 cells (Figure 5A) and at 10 uM in Jurkat cells (Figure 5B). A kinetic analysis of TQ was then conducted on Bcl-2 expression in HCT-116 cells, using 400 uM TQ (Figure 5C) and 200 uM TQ in Jurkat cells (Figure 5D), concentrations at which Bcl-2 became undetectable after 24 h of treatment in HCT-116 cells (Figure 5A) and Jurkat cells (Figure 5B).