*6.1.1. Intrinsic pathway*

SHetA2 is shown to selectively induce apoptosis in various cancer cell lines by targeting the mitochondria [32]. Exposure of SHetA2 to squamous carcinoma cells resulted in a decrease in mitochondrial permeability transition, followed by the release of cytochrome *c* into the cytoplasm, activation of caspase-3, and the induction of intrinsic apoptotic pathway. Further studies proposed that SHetA2-mediated mitochondrial swelling involved lowering the levels of anti-apoptotic proteins such as Bcl-XL and Bcl-2 in A2780 ovarian cancer cell [9]. However, the pro-apoptotic Bax expression was unaffected by SHetA2, suggesting SHetA2 may regulate the levels of Bcl-2 to promote apoptosis [9]. On the other hand, exposure of SHetA2 to normal ovarian or endometrial cells was found to increase both Bcl-XL and Bcl-2 protein levels. The up-regulation of these anti-apoptotic proteins may provide the cytoprotective effects necessary to block SHetA2-induced apoptosis in normal cells [9].

**Figure 7.** Pathways involving SHetA2-induced apoptosis.

#### *6.1.2. Extrinsic pathway (death receptor pathway)*

**6. Mechanism of action**

80 Anti-cancer Drugs - Nature, Synthesis and Cell

**6.1. Induction of apoptosis**

*6.1.1. Intrinsic pathway*

growth, differentiation, and angiogenesis.

to block SHetA2-induced apoptosis in normal cells [9].

**Figure 7.** Pathways involving SHetA2-induced apoptosis.

The biological targets and SHetA2's mechanism of action have been extensively studied. Coimmunoprecipitation experiments demonstrated that SHetA2 interfered with the binding of mortalin, a molecular chaperone to p53 and p66 Src homologous-collagen homolog (p66shc) in A2780 ovarian cancer cell line, leading to mitochondrial swelling and mitophagy and finally apoptosis [8]. SHetA2 is also involved in modulating other cellular processes including cell-

SHetA2 is shown to selectively induce apoptosis in various cancer cell lines by targeting the mitochondria [32]. Exposure of SHetA2 to squamous carcinoma cells resulted in a decrease in mitochondrial permeability transition, followed by the release of cytochrome *c* into the cytoplasm, activation of caspase-3, and the induction of intrinsic apoptotic pathway. Further studies proposed that SHetA2-mediated mitochondrial swelling involved lowering the levels of anti-apoptotic proteins such as Bcl-XL and Bcl-2 in A2780 ovarian cancer cell [9]. However, the pro-apoptotic Bax expression was unaffected by SHetA2, suggesting SHetA2 may regulate the levels of Bcl-2 to promote apoptosis [9]. On the other hand, exposure of SHetA2 to normal ovarian or endometrial cells was found to increase both Bcl-XL and Bcl-2 protein levels. The up-regulation of these anti-apoptotic proteins may provide the cytoprotective effects necessary Exposure of non-small cell lung cancer cells (NSCLC) to SHetA2 revealed the induction of the extrinsic apoptotic pathway involving the death receptor 5 (DR5) [49]. It was shown that SHetA2-enhanced DR5 expression through the enhanced binding of CAAT/enhancer-binding protein homologous protein (CHOP) to its binding site located in the 5'-flanking region of the DR5 gene. Since CHOP is highly inducible during endoplasmic reticulum (ER) stress, this finding suggests that SHetA2 may act as an inducer of ER stress. The induction of DR5 expression leads to caspase 8-dependent apoptosis. Moreover, the induction of DR5 was shown to enhance tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis (**Figure 7**). Further studies found that down-regulation of cellular FLICE-inhibitory protein (c-FLIP), and to a lesser extent of survivin, were involved in SHetA2-induced apoptosis, as well as enhancement of TRAIL-initiated apoptosis [50]. c-FLIP is a major inhibitor of the extrinsic apoptotic pathway [51], while survivin modulates both intrinsic and extrinsic apoptotic pathways [52].

**Figure 8.** Working model for the apoptotic pathway involving NF-kB in ovarian cancer cells.

TRAIL and its receptors have also attracted much attention recently because TRAIL preferentially induces apoptosis in malignant cells while sparing most normal cells [53]. SHetA2 was found to sensitize ovarian carcinomas that are resistant to these DR ligands, without harming the normal cells [54, 55]. This suggests a possible synergistic effect Further investigation identified that downregulation of NF-kB transactivation activity by SHetA2 was involved in this sensitizing effect, triggering extrinsic apoptosis in cancer cells resistant to certain chemotherapeutics [55]. SHetA2 was shown to repress the upstream IkB kinase (IKK) activity that resulted in NF-kB downregulation. Apart from DR ligands, SHetA2 has also shown its chemosensitizing effects against cisplatin-resistant ovarian cancer through p53-independent pathways [6, 56]. However, such enhancement of sensitivities was not observed in resistant uterine cancer cell lines for a number of chemotherapeutics in combination with SHetA2 treatment (**Figure 8**) [57].


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

#### *6.1.3. ROS and GSH*

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 mitochondrial swelling and the release of cytochrome *c* in various cancer cell lines [6, 32]. Studies have also shown that SHetA2 is able to form adducts with glutathione (GSH) [7, 58]. This was reflected by the generation of ROS as reported in several pharmacokinetic studies [58, 59]. GSH is essential for cell survival. Adduct formation with GSH causes GSH depletion, leading to accumulation of ROS resulting in oxidative mitochondrial damage, ultimately causing cell death [60]. Although the exact role of GSH depletion in apoptosis is still controversial, it remains an early hallmark in the progression of cell death in numerous cell types [61]. Therefore, adduct formation between SHetA2 and GSH could be vital to the induction of apoptosis, as indicated by the ROS generation. However, further studies suggested that ROS generation appeared to be a consequence of, and not a cause for, mitochondrial swelling and apoptosis induced by SHetA2 treatment [7, 9]. Addition of GSH did not attenuate SHetA2 induced apoptosis [55]. It was also noted that the cellular GSH level was in the μM range, while μM SHetA2 is sufficient to kill cells, indicating that GSH depletion and ROS accumulation are not the only mechanisms of action. These findings suggest that SHetA2 probably acts through several mechanisms of action to bring about its apoptotic effect, along with other anticancer activities, such as cell cycle arrest and induction of differentiation (**Table 4**).

## **6.2. Induction of cell-cycle arrest and differentiation**

Apart from apoptosis, SHetA2 repression of NF-kB expression also initiates a series of events that can lead to cell-cycle arrest and cell differentiation. SHetA2-induced Cyclin D1 degradation in both Caki-1 renal cancer and normal HK-2 cell lines results in the accumulation of cells in the G0-G1 phase [7]. Cyclin D1 degradation alone has shown to be sufficient in inducing G1 cell cycle arrest [11]. Cyclin D1 transcription can be induced by NF-kB through multiple NFkB-binding sites in the Cyclin D1 promoter, which is consistent with the observed down regulation of Cyclin D1. Also, SHetA2 can induce cellular differentiation in kidney [7], as well as in ovarian cancer organotypic cultures and xenografts [5]. Treatment of these cancer cells shows that 1 μM SHetA2 is sufficient to reverse the cancerous phenotype depending on the status of the cells. However, at higher concentrations of SHetA2, apoptosis would dominate over differentiation. The mechanism for differentiation induction is likely to be associated with the upregulation of E-Cadherin in renal cancer cells observed with the repression of NF-kB [7]. Decrease or loss of nuclear E-Cadherin expression is associated with poor prognosis in kidney cancer, as it confers the ability to migrate and invade [64]. Therefore, up-regulation or restoration of E-Cadherin's function has been one of the therapeutic goals for anticancer treatments.
