**6.3. Antiangiogenic activity**

uterine cancer cell lines for a number of chemotherapeutics in combination with SHetA2

Intrinsic ↓ GSH Redox regulation [7]

Extrinsic ↑ Caspase-8 Proapoptotic [10]

Possible biological targets Effect Reference

*↓ Bcl-2* Antiapoptotic [9] *↓ Bcl-XL* Antiapoptotic [9] ↓ Survivin Antiapoptotic [50] ↑ Caspase-9 Proapoptotic [49] ↑ Caspase-3 Proapoptotic [32] ↑ Bid Proapoptotic [49]

↑ Bip/GRP78, IRE1α, ATF4, XBP1 ER stress [49] ↑ DR-5 Cell death [55] ↑ CHOP Cell death [49] ↓ IKK Cell survival [55] ↓ NF-kB Cell survival [50] ↓ c-FLIP Antiapoptotic [50]

↓ Cyclin D1 G1 arrest [11] ↓ AP-1 Cell-cycle progression [62]

↑ E-Cadherin Differentiation [7] ↑ MUC1 Differentiation [44]

↑ Thrombospondin (TSP-4) Antiangiogenic [12] ↓ Thymidine Phosphorylase (TP) Angiogenic [12] ↓ VEGF Angiogenic [12]

↓ KIT kinase (c-KIT) Cell death [63]

Increased reactive oxygen species (ROS) level was initially thought to be responsible for SHetA2-induced apoptosis. Multiple studies have documented the ROS generation along with

**Table 4.** Possible biological pathways affected by SHetA2 and its anticancer activities.

treatment (**Figure 8**) [57].

82 Anti-cancer Drugs - Nature, Synthesis and Cell

Cell cycle pathways

Differentiation induction pathways

Antiangiogenic pathways

Signal transduction pathways

*6.1.3. ROS and GSH*

**Apoptotic pathways affected by SHetA2**

Microarray analysis have suggested that SHetA2 treatment results in significant up-regulation of thrombospondin-4 (TSP-4), and down-regulation of thymidine phosphorylase (TP) in A2780 ovarian cancer cells. TSP-4 and TP protein levels followed those of the mRNA. TSP-4 was hypothesized to be antiangiogenic due to the presence of type-III repeats which is also found in TSP-1. The type-III repeats in TSP-1 has been reported to inhibit the binding of fibroblast growth factor-2 to endothelial cells, a process that leads to endothelial cell proliferation *in vitro* [65]. As for TP, it has found to be angiogenic through its ability to convert thymidine to thymine and the angiogenic 2-deoxy-D-ribose-1-phosphate, as well as its metabolite, deoxy-D-ribose. Protein levels of the angiogenic vascular endothelial growth factor A (VEGF) was also found to be downregulated despite its up-regulated mRNA level upon SHetA2 treatment. Interestingly, both mRNA and protein levels of the angiogenic basic fibroblast growth factor (bFGF) were also up-regulated, but its effect was deemed limited due to brief up-regulation of the gene only after hours of treatment with high concentration of SHetA2 [12]. Hence, the net effect of SHetA2 on these various proteins is antiangiogenic, and is supported by the decrease of endothelial tube formation observed in a number of cancer cell lines as well as human umbilical vascular endothelial cells (HUVECs) [12].

**Figure 9.** Structural comparison of SHetA2 and its metabolite with sorafenib and linifanib.

#### **6.4. Kinase inhibitory activity**

SHetA2 is also being evaluated for its ability to inhibit kinase activity based on its structural similarity to sorafenib and linifanib (**Figure 9**) [63]. Sorafenib is an FDA approved diarylurea multikinase inhibitor that inhibits tumor growth, while linifanib is a KIT-3 kinase inhibitor [66, 67]. All three compounds consist of a three atom urea or thiourea linker between two aromatic rings, of which this structural conformation is found to be vital for the formation of key Hbonds within the binding pockets of several kinases, including B-Raf, BCR-ABL, and KIT [63]. Upon evaluation with 442 different human kinases, SHetA2(**Figure 9**) has exhibited good binding affinity for KIT kinase (binding constant, Kd = 820 nM). This indicates that SHetA2 is a potential candidate for kinase inhibitor development. More importantly, one of the metabolites of SHetA2 (**Figure 9**, **25**) has also shown comparable binding affinity for KIT kinase (Kd = 1200 nM) [63]. This suggests that other metabolites of SHetA2 may also be active, which could have acted via different mechanisms of action and contributed to the various anticancer effects observed, making it a versatile chemotherapeutic agent.
