Dysregulation of the programmed cell death process (apoptosis) is a hallmark of many types of malignancies and the cause of cancer cell resistance to chemotherapy 
. Thus restoring apoptosis is a promising strategy for effectively treating cancers 
. Currently, many therapeutic approaches, including various biologicals, hormones, and small molecule chemotherapeutic agents such as ABT737, AT101, HS-113 and chloroquine, inhibit tumors by triggering cell apoptosis 
. Previous studies have demonstrated that some benzothiazole derivatives can induce apoptosis 
, but the mechanism has not been clearly determined. In the present study, our results show that YLT322 is a broad spectrum anti-cancer compound with high apoptotic-inducing activity to a panel of cancer cells with the most potent effect observed with HepG2. Moreover, the anti-cancer effect of YLT322 was demonstrated to be associated with the induction of apoptosis.
Possible drug targets for regulating cell apoptosis have been widely studied 
. In general, the action of apoptosis is through two very distinct signaling pathways: intrinsic pathway or extrinsic pathway 
. Caspase-3, the major effector caspase, is responsible for the cleavage of the majority of polypeptides that undergo proteolysis in an apoptotic cell and plays a central role in both types of apoptotic pathways 
. It is activated by upstream effector proteins including caspase-8 and caspase-9, the apical proteases in the extrinsic and intrinsic pathways, respectively 
. After YLT322 treatment, we observed in cells a reduction in procaspase-3, -8 and -9 and a concomitant increase in their cleavage, indicative of enzyme activation,. Addition of the irreversible inhibitors, Z-VAD-FMK (caspase inhibitors) and Ac-LETD-FMK(caspase-9 inhibitors) significantly decreased the percentage of apoptotic cells after treatment with YLT322, suggesting that cell apoptosis induced by YLT322 may be dependent on caspase activation. However, Ac-IETD-FMK, a caspase-8 inhibitor, was unable to antagonize YLT322-induced cell death. It is possible that caspase-8 plays a non-apoptotic role in the cell, such as regulating cell differentiation and senescence 
. We assumed that the activation of caspase-8 observed after YLT322 treatment is a possible downstream “bystander” event which has been reported elsewhere 
. These data suggest that the intrinsic apoptosis pathway is more important than the extrinsic pathway in apoptosis induced by YLT322.
A key step in the intrinsic apoptotic pathway is the disruption of the mitochondrial membrane, which triggers the release of cytochrome c from mitochondria to cytosol. From our study, a reduction in ΔΨ and a release of cytochrome c from the mitochondria into the cytosol were observed in a dose-dependent manner after YLT322 treatment.
Bcl-2 protein family plays an important role in the regulation of mitochondrial apoptosis pathway 
. Multidomain pro-apoptotic proteins such as Bax and Bid can open the ion channel in the outer mitochondrial membrane in the presence of a death signal, leading to mitochondrial membrane permeabilization and release of cytochrome c
into the cytosol 
. Cytochrome c
in the cytosol binds to the scaffolding protein Apaf-1 and pro-caspase-9 to form the apoptosome, which triggers the apoptotic cascade reaction thereafter. The anti-apoptotic Bcl-2 family members such as Bcl-2 and Bcl-xl inhibit cytochrome c
release by blocking the activation of pro-apoptotic proteins. For most cancers, the over-expression of Bcl-2 protein associates with poor survival, uncontrolled progression and resistance to anticancer agents, making Bcl-2 family members promising anticancer drug targets 
. Our results showed that the expression of Bcl-2 decreased in cells treated with YLT322, whereas that of Bax increased. We assumed that cytochrome c
release from the mitochondria into the cytosol upon YLT322 treatment is due to the down-regulation of Bcl-2 and/or up-regulation of Bax.
Studies have suggested that Akt/p44/42 MAPK pathway plays essential roles in apoptosis and could be modulated for cancer prevention and treatment 
. Here, we found that treatment with YLT322 decreased the phosphorylation of both Akt and p44/42 MAPK. This suggests that Akt and p44/42 MAPK may contribute to the apoptosis induced by YLT322.
YLT322 treatment on nude mice bearing tumors resulted in significant inhibition of tumor volume without any apparent sign of toxicity. Immunohistochemical studies revealed that the regression of tumor size in mice models by YLT322 was also associated with the activation of apoptosis, as demonstrated by of the presence of active caspase-3-positive and TUNEL-positive cells in tumor xenograft samples. YLT322 also caused a decrease in Ki67-positive cells, suggesting a reduction in cell proliferation.
In conclusion, our study demonstrated that YLT322 inhibits cell growth/proliferation by inducing apoptosis via the intrinsic pathway. In addition, YLT322 suppressed the growth of human liver cancer xenografts without significant toxicity, suggesting that it may be a potential candidate for cancer therapy. In depth studies are still needed to elucidate the precise targets mediating the anti-cancer effects of YLT322.