## 4 Discussion  
The overexpression of UHRF1, detected in many solid and hematological tumors, is known to inhibit the expression of various tumor suppressor genes (TSGs) and to inhibit apoptosis [4; 7; 17]. Several studies have shown that natural anticancer compounds, including TQ, can decrease UHRF1 expression in cancer cells and reactivate silenced TSGs with the subsequent induction of apoptosis [1; 4; 5; 11; 17; 44]. Recently, TQ was shown to induce the fast auto-polyubiquitin of UHRF1 through its RING domain, and this effect was correlated with a decrease in the expression of the HAUSP deubiquitininase [11]. In the present study, two natural compounds--TQ and doxorubicin, which exhibit anticancer properties on various cancer cells through the targeting of several signaling pathways--were evaluated for their effects on UHRF1 expression in colon cancer cells and Jurkat cells. The kinetic effects of TQ on UHRF1 expression in colon cancer and Jurkat cells showed that UHRF1 was ubiquitinated, as reflected by the appearance of high-molecular-weight bands, and this effect was associated with a downregulation of HAUSP. Like the TQ effects, doxorubicin also induced a dose-dependent degradation of UHRF1 in colon cancer cells and Jurkat cells. However, the kinetic effect of doxorubicin on UHRF1 expression did not show the appearance of any high-molecular-weight bands of UHRF1 in either of the cancer cell lines, suggesting that UHRF1 ubiquitination in cancer cells is triggered only by TQ, and not by doxorubicin. The present findings support the presence of a strong correlation between the TQ-induced ubiquitination of UHRF1 and its subsequent degradation in cancer cells [11]. In the present study, the time-course effects of doxorubicin on UHRF1 expression in colon cancer cells and Jurkat cells did not reveal any high-molecular-weight bands in either cancer cell line. This suggests that the degradation of UHRF1 in response to TQ requires auto-ubiquitination through its RING domain, but this step is not required in the response to other natural compounds that exhibit anticancer activities. This process could involve, in part, the downregulation of HAUSP to allow the auto-ubiquitination of UHRF1 and its degradation, followed by the subsequent upregulation of TSGs, the inhibition of cell  
Figure 5: Dose- and time-dependent effects of TQ on the protein expression of Bcl-2 in cancer cells. HCT-116 cells were exposed to increasing concentrations of TQ for 24 h (**A**) or exposed to 400 \(\upmu\)M TQ for different times (**C**). Jurkat cells were exposed to increasing concentrations of TQ for 24 h (**B**) or exposed to 200 \(\upmu\)M TQ for different times (**D**). Expres-sion of Bcl-2 was analyzed via Western blotting, as described in the Materials and Methods. proliferation and the induction of apoptosis. Indeed, several natural products, including naphthalene [19], shikonin [20], curcumin [5], EGCG [4], resveratrol [21] and luteolin [22], as well as TQ [1; 25], have been shown to decrease the expression of UHRF1 in cancer cells, but their underlying molecular mechanisms are largely unknown. Few studies have conducted kinetic analyses of the effects of these natural products on UHRF1 expression. The time-course effects of Antho 50, an anthocyanin-rich dietary bilberry extract, on UHRF1 expression in cells from patients with B-cell chronic lymphocytic leukemia (B CLL) showed a progressive decrease in UHRF1 expression, but without the appearance of high-molecular-weight bands of UHRF1 [45]. In the present study, the time-course effects of TQ on UHRF1 in colon cancer, as a model of solid tumors, and in Jurkat cells, as a hematological tumor model, revealed that TQ induced the appearance of high-molecular-weight bands of UHRF1, but doxorubicin did not. This finding suggests that TQ can induce UHRF1 ubiquitination in cancer cells as an indispensable event that triggers the onset of apoptosis, and this process could be as a result of the disassociation between HAUSP and UHRF1.