## 4 Anti-Cancer Activities of Tanshinones
### Pro-Differentiation of Cancer Cells  
Aside from anti-proliferation and pro-apoptosis, induction of differentiation is another important mechanism to suppress cancer, as de-differentiation is a hallmark of cancer. Pharmacological induction of terminal differentiation has already been used to treat leukemia with all-trans retinoic acid (ATRA, a pro-differentiation drug) and arsenic-base agents [195; 196; 197]. Induction of differentiation by TIIA has been documented in several studies. In 1997, [PERSON] _et al._[198] investigated the differentiation effect of TIIA on leukemia cells, and found that after 4 days of TIIA treatment (1.7 \(\upmu\)M), more than half of HL-60 human leukemia cells were induced into mature neutrophils, in which metamyelocytes were 46%, and banded and segmented neutrophils were 12%, respectively. The HL-60 cell growth was inhibited, accompanied by G\({}_{0}\)/G\({}_{1}\) arrest, decreased c-Myc and increased c-Fos. Similar results were obtained from later studies [199; 200; 201]. [PERSON] _et al._[199] reported that after five days of TIIA treatment (1.7 \(\upmu\)M), the majority of leukemia NB4 cells (91.3%) converted into more differentiated cells, including 26% of myelocytes and metamyelocytes as well as 68% of band form and segmented neutrophils. The induced expression of membrane cluster differentiation (CD) antigens was consistent with the differentiation fates, _i.e._, CD33 (a marker of myeloid cells) was decreased and CD11b (a marker of terminal differentiation) was increased. ATRA-resistant leukemia cells could also respond to TIIA. After 4 days of incubation with 3.4 \(\upmu\)M of TIIA, ATRA-resistant leukemia MR-2 cells underwent morphological and functional differentiation as indicated by small cell bulk, decreased nucleus/cytoplasm ratio, rough chromatin, disappearance of nucleolus, formation of anomalous nucleus, appearance of metamyelocyte, decrease of CD33 and increase of CD11b, and reduction of nitroblue tetrazolium test (NBT reduction, a marker of functional differentiation) [202]. Using acute promyelocytic leukemia (APL) cells isolated from patients, [PERSON] _et al._[203] tested the differentiation effect of TIIA on primary leukemia cells. After incubation with 1.7 \(\upmu\)M of TIIA for seven days, morphological and functional differentiation was observed, accompanied by G\({}_{0}\)/G\({}_{1}\) arrest and growth inhibition. In addition, a synergistic effect of TIIA and ATRA (1.7 \(\upmu\)M of TIIA plus 0.4, 0.8 or 1.6 \(\upmu\)M of ATRA, respectively) on cell differentiation was reported in leukemia NB4 cells. The growth inhibition rate, differentiation rate, NBT reduction,differentiation markers CD33 (decrease) and CD11b (increase) were much higher in the combination group than ATRA or TIIA alone [204]. Moreover, [PERSON] _et al._[173] found that C/EBP\(\beta\)/CHOP ratio might be a determinant of TIIA-induced differentiation response of leukemia cells. C/EBP\(\beta\) and CHOP (also known as GADD153) are members of the C/EBP family of leucine zipper transcription factors which interact with specific DNA sequences and affect the transcription of proliferation- and differentiation-related genes [205]. Expression of C/EBP\(\beta\) drives immature granulocytes to enter into terminal differentiation [206], yet expression of CHOP reverses this differentiation direction through inhibiting C/EBP\(\beta\) binding to its target DNA [207]. TIIA at 0.34-3.4 \(\upmu\)M induced leukemia NB4 and MR-2 cell differentiation as indicated by increased expression of CD11b, which was associated with up-regulation of C/EBP\(\beta\) expression and increased C/EBP\(\beta\)/CHOP ratio [173]. However, higher concentration of TIIA (34 \(\upmu\)M) showed less pro-differentiation effect, along with up-regulation of CHOP expression but a decrease of C/EBP\(\beta\)/CHOP ratio. Very likely the onset of apoptosis subverted the differentiation program at the higher exposure concentration.