Anti parasitic drug Albendazole Exhibits Anti-Neoplastic Actions against Gastric Cancer Cells

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Abstract​

Albendazole (ABZ) has been reported to display anti-tumoral actions against various maliganncies, but possible impact of ABZ on gastric cancer has not been deciphered. As aberrant phosphorylation of STAT3 and STAT5 proteins can regulate the growth and progression of gastric cancer, we postulated that ABZ may interrupt the activation of these oncogenic transcription factors. We found that ABZ exposure abrogated STAT3/5 activation, inhibited phosphorylation of Janus-activated kinases 1/2 and Src and enhanced the levels of SHP-1 protein. Silencing of SHP-1 gene by small interfering RNA (siRNA) reversed the ABZ-promoted attenuation of STAT3 as well as STAT5 activation and cellular apoptosis. In addition, these effects were noted to be driven by an augmented levels of reactive oxygen species caused by drug-induced GSH/GSSG imbalance. Thus, the data indicates that ABZ can modulate the activation of STAT3 and STAT5 by pleiotropic mechanisms in gastric cancer cells.

 

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STAT3 and STAT5 promote tumor progression by regulating the expression of cell cycle, survival and pro-inflammatory genes. In addition, they control mitochondrial functions, metabolism and stemness, as discussed below (Figure 2). Mechanisms of tumorigenic activity of STAT3 and STAT5 signaling in solid tumors.

https://www.ncbi.nlm.nih.gov › pmc

STAT3 and STAT5 Activation in Solid Cancers - PMC - NCBI​

 

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Introduction​

Signal transducers and activators of transcription (STAT) family consists of seven diverse members, named STAT1 to STAT6, STAT5a, and STAT5b [1,2]. Accumulated evidences have indicated that STAT members can exhibit diverse actions under both physiological and pathological conditions [3,4,5,6,7]. Of the STAT family members, STAT3 and STAT5 can regulate various hallmarks that are closely associated with tumorigenesis [8,9,10,11,12,13]. Phosphorylation of STATs can be driven via the stimulation of Janus-activated kinases (JAK) and Src families of proteins [14,15,16]. The activation can promote their homodimerization, movement into the nucleus, DNA binding, thus leading to transcription of oncogenic genes [4,17,18]. Interestingly, protein tyrosine phosphatases (PTPs) such as SHP-1, SHP-2, PTPε, and PTEN can negatively regulate the STAT signaling pathway [19,20,21]. In addition, various previous reports have indicated that a number of naturally derived pharmacological agents can attenuate STAT activation, which may have a great potential in the prevention and therapy of cancer [13,22,23,24,25,26].

Gastric cancer remains a lethal disease [25,27,28,29]. It has emerged as a significant health problem characterized by poor prognosis and disease relapse [30,31,32]. Gastric cancer has been treated using different strategies such as surgery, chemotherapy, and radiation therapy, but the clinical outcome has been not quite effective with development chemoresistance being a major obstacle [31,33,34,35,36,37]. Hence, elucidation of the biological characteristics and molecular mechanisms of novel agents may be beneficial for the gastric cancer treatment. As the abnormal expression and dysregulation of STAT3/5 have been reported in various malignancies including gastric cancer [38], targeting of STAT3/5 phosphorylation may be beneficial for gastric cancer treatment.

A number of agents isolated from medicinal plants have been used for treatment against diverse malignancies [39,40,41,42]. Albendazole (Methyl 5-(propylthio)-2-benzimidazolecarbonate)) carbamate, ABZ), a well-known benzimidazole derivative carbamate anthelminthic [43], can target helminth cell proliferation through disputing microtubule assembly and affecting glucose uptake [44,45]. ABZ has been reported to exhibit diverse anticancer actions against several tumor cell lines such as hepatocellular carcinoma, colorectal cancer, non-small cell lung cancer, as well as cutaneous squamous cell carcinoma [46,47,48,49]. For instance, ABZ was reported to be effective against non-small cell lung cancer cells by reducing both vascular endothelial growth factor (VEGF and hypoxia-inducible factor-1-α (HIF-1-α) activities [48]. It was also found to promote apoptosis in human leukemia cells by causing TNF-α upregulation [50]. However, the influence of ABZ on gastric cancer cells and the underlying mechanisms have not been evaluated previously. In this study, it was investigated whether ABZ can impact the activation of STAT3 and STAT5 pathway in gastric cancer cells and thereby exhibit pleiotropic actions on tumor progression as well as survival.

 

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3. Results​

3.1. ABZ Suppresses the Cell Viability​

The structure of ABZ is shown in Figure 1. The cytotoxic actions of ABZ against SNU-16 and SNU-1 and normal gastric GES-1 cells was evaluated by MTT assay. We observed that ABZ displayed higher cytotoxic effects against SNU-16 and SNU-1 as compared to GES-1 cells, thereby suggesting its selectivity against tumor cells (Figure 2A).

 

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3. Results​

3.1. ABZ Suppresses the Cell Viability​

The structure of ABZ is shown in Figure 1. The cytotoxic actions of ABZ against SNU-16 and SNU-1 and normal gastric GES-1 cells was evaluated by MTT assay. We observed that ABZ displayed higher cytotoxic effects against SNU-16 and SNU-1 as compared to GES-1 cells, thereby suggesting its selectivity against tumor cells (Figure 2A).

2.2. Cell Lines and Culture Conditions​

SNU-1 and SNU-16 cells were obtained from the Korean Cell Line Bank (Seoul, Korea). Human normal gastric epithelia mucosa GES-1 cells were provided by Dr. Sang-kil Lee (Division of Gastroenterology, Department of Internal Medicine, Institute of Gastroenterology, Yonsei University College of Medicine, Seoul, Korea). SNU-1, SNU-16, and GES-1 cells were cultured in RPMI-1640 medium containing 10% FBS.

 

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3.2. ABZ Substantially Affected STAT3 and STAT5 Phosphorylation​

In order to identify if ABZ can alter STAT3/5 activity, SNU-16 and SNU-1 cells were treated with different doses of drug for 3 h or exposed to different time periods at 50 µM ABZ concentration. As shown in Figure 2B,C, constitutive STAT3 and STAT5 phosphorylation was substantially suppressed upon ABZ exposure. Moreover, as depicted in Figure 2D,E, ABZ could markedly attenuate the DNA-binding activities of STAT proteins. Additionally, as shown in Figure 2F,G, ABZ also effectively reduced the nuclear translocation of STAT3 and STAT5 proteins thus preventing the gene transcription.

3.3. ABZ Alters Phosphorylation of JAK1/2 and Src Kinases​

We next deciphered if ABZ can alter the levels of kinases involved in regulating activation of STATs. As shown in Figure 3A,B, ABZ substantially downregulated the activation of JAK1, JAK2 and Src kinases in a concentration and time-controlled fashion in SNU-16 and SNU-1 cells.

 

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3.4. Tyrosine Phosphatases Affect STAT3/5 Modulatory Actions of ABZ​

To analyze the mechanism of ABZ-stimulated inhibition of STATs phosphorylation, we examined the effect of ABZ in modulating the levels of protein tyrosine phosphatases (PTPs). It was noted that the exposure to sodium pervanadate reversed the attenuation of STAT3 and STAT5 phosphorylation, highlighting that this activity of ABZ can be regulated by a tyrosine phosphatase (Figure 3C). We next observed that ABZ only induced SHP-1 expression but did not affect other PTPs (SHP-2, PTPε, PTEN) in both SNU-16 and SNU-1 cells. ABZ also augmented the mRNA levels of SHP-1 (Figure 3E). Additionally, upon SHP-1 knockdown, SHP-1 expression was substantially reduced and ABZ was unable to affect STAT3 and STAT5 phosphorylation in SHP-1 knocked down cells (Figure 3F).

3.5. ABZ Alters the Levels of Various Oncogenic Proteins and Caused Apoptosis​

SNU-16 and SNU-1 cells exposed to 50 µM of ABZ and western blotting for apoptotic markers was done. As shown in Figure 4A, ABZ promoted apoptosis by increasing the breakdown of caspase-3 and PARP proteins. ABZ also downregulated the expression of diverse proteins involved in regulating various hallmarks of tumor growth at protein and mRNA levels (Figure 4B,C). As shown in Figure 4D, the deletion of SHP-1 also attenuated ABZ-induced PARP cleavage which suggested that SHP-1 may play a vital role in regulating anti-cancer properties of ABZ.

 

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3.7. ABZ Exhibits Anti-Neoplastic Effect through ROS-Mediated Events​

To explore the potential involvement of ROS in observed anti-cancer functions of ABZ, we studied the impact of ABZ on the GSH/GSSG system. As demonstrated in Figure 6A, ABZ decreased GSH levels in SNU-16 and SNU-1 cells. On the contrary, GSSG and GSSG/SGH ratio was noted to be increased. Additionally, to validate if ABZ can generate oxidative stress, ROS levels were quantitated through H2DCF-DA staining. A significant increase in ROS levels were noted upon ABZ exposure and antioxidant NAC prevented ABZ-induced ROS production (Figure 6B). Interestingly, pretreatment of NAC only partially suppressed SHP-1 induction and abolished STAT3 (SNU-16 and SNU-1 cells) and STAT5 (SNU-16 cells) suppression caused by ABZ treatment (Figure 6C). Furthermore, antioxidant pretreatment could also substantially mitigate apoptosis promoted by ABZ, thereby demonstrating that oxidative stress can regulate pro-apoptotic actions of ABZ (Figure 6D,E).

 

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3.6. ABZ Induces Apoptotic Cell Death in Gastric Cancer Cells​

The impact of ABZ on apoptosis was studied using cell cycle analysis, annexin and TUNEL assays. Early apoptotic cells are annexin V-FITC+/PI-, whereas late apoptotic cells are annexin V-FITC+/PI+. As shown in Figure 5A, ABZ enhanced aggregation of cell population in sub G1 phase. And ABZ produced late apoptosis (annexin V-FITC+/PI+) as evidenced by shifting of peak to right side (Figure 5B,C). The results of live and dead assay also confirmed that ABZ also significantly attenuated viability of gastric cancer cells (Figure 5D).

 

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4. Discussions​

Previous reports have indicated that ABZ may display significant anti-tumor activities in different tumor models, with gastric cancer being an exception [48,58]. The purpose here was to elucidate the anti-cancer impact of ABZ and also to unravel its mode of actions. We found that ABZ targeted both STAT3 and STAT5 activation, up-regulated the induction of SHP-1 protein and promoted ROS accumulation which can lead to substantial apoptosis (Figure 7).