PTEN Activation by DNA Damage Induces Protective Autophagy in Response to Cucurbitacin B in Hepatocellular Carcinoma Cells

Cucurbitacin B (Cuc B), a natural product, induced both protective autophagy and DNA damage mediated by ROS while the detailed mechanisms remain unclear. This study explored the mechanism of Cuc B-induced DNA damage and autophagy. Cuc B decreased cell viability in concentration- and time-dependent manners. Cuc B caused long comet tails and increased expression of γ-H2AX, phosphorylation of ATM/ATR, and Chk1/Chk2. Cuc B induced autophagy as evidenced by monodansylcadaverine (MDC) staining, increased expression of LC3II, phosphorylated ULK1, and decreased expression of phosphorylated AKT, mTOR. Cuc B induced apoptosis mediated by Bcl-2 family proteins and caspase activation. Furthermore, Cuc B induced ROS formation, which was inhibited by N-acetyl-L-cysteine (NAC). NAC pretreatment dramatically reversed Cuc B-induced DNA damage, autophagy, and apoptosis. Cuc B-induced apoptosis was reversed by NAC but enhanced by 3-methyladenine (3-MA), chloroquine (CQ), and silencing phosphatase and tensin homolog (PTEN). 3-MA and CQ showed no effect on Cuc B-induced DNA damage. In addition, Cuc B increased PTEN phosphorylation and silence PTEN restored Cuc B-induced autophagic protein expressions without affecting DNA damage. In summary, Cuc B induced DNA damage, apoptosis, and protective autophagy mediated by ROS. PTEN activation in response to DNA damage bridged DNA damage and prosurvival autophagy.


Introduction
Programmed cell death (PCD), a process carried out in a regulated manner, ubiquitously occurs throughout most multicellular organisms' lifespan. To date, three major types of PCD, distinct both morphologically and biochemically, have been established: apoptosis (type I cell death), autophagic cell death (type II), and regulated necrosis (type III) [1][2][3]. The first and widely investigated type of PCD is apoptosis. Apoptosis is triggered by the activation of cell-surface death receptors by their ligands (the extrinsic pathway) or by induction of the permeabilization of the mitochondrial outer membrane through the Bcl-2 family proapoptotic proteins (Bax, Bak, etc.) (the intrinsic pathway) [1,4]. Autophagy, a stress response to starvation, acts as an important homeostatic cellular recycling mechanism responsible for degrading unnecessary or dysfunctional cellular organelles and proteins in living cells [5]. Autophagy is characterized by the appearance of large intracellular vesicles and finely controlled by the Atg (autophagy-related gene) family of proteins. In general, it represents a failed attempt to overcome lethal stress and serve as a prosurvival process in response to various stresses. Thus, its function as an active cell death mechanism remains controversial [1]. Actually, most reported autophagy induced by natural products was prosurvival [6,7]. Regulated necrosis is morphologically characterized by cytoplasmic granulation, organelle and/or cellular swelling resulting from cellular leakage [8]. 2 Oxidative Medicine and Cellular Longevity Accumulated evidence showed that though apoptosis and autophagy were executed through distinct signaling pathways, overlapping signals were engaged in response to specific stimuli [1]. This crosstalk could be mediated by the interactions between Beclin-1 and Bcl-2/Bcl-xL and between FADD and Atg5, caspase-and calpain-mediated cleavage of autophagy-related proteins, and autophagic degradation of caspases [9][10][11][12][13]. Reactive oxygen species (ROS) plays important roles in mediating apoptosis and autophagy in response to a panel of natural products such as evodiamine [14], oridonin [15], graveoline [16], total tanshinones [17], and erianin [18].

Cell
Culture. Hepatocellular carcinoma BEL-7402 cells gifted by Professor Jian Ding from the Shanghai Institute of Materia Medica (Shanghai, China) were maintained in a monolayer culture in 95% air and 5% CO 2 at 37 ∘ C in RPMI Medium 1640 (Gibco) supplemented with 10% FBS (Gibco), 100 U/mL penicillin, and 100 g/mL streptomycin (Invitrogen). LDH release assay was determined by LDH-cytotoxicity assay kit (Beyotime, China) according to the manufacturer's instructions.

Colony Formation
Assay. Cells seeded in 6-well plates were treated with or without Cuc B (5-20 nM) and the colony formation assay was performed as in our previous report [27].

Comet Assay.
Comet assay was performed as in previous report with minor revisions [37]. Briefly, microscopic slides were coated with 1% normal agarose (GE Healthcare) followed by adding 1% low melting point (LMP) agarose onto each slide and then covering slides with coverslips. The cell suspensions mixed with 1% LMP agarose (1 : 1) were pipetted onto agarose-coated slips. After cooling down, the coverslips were removed and the slides were lowered into freshly made prechilled lysis buffer (2.5 M NaCl, 100 mM EDTA, 10 mM Tris, 1% Triton X-100, and pH 10) for 1 h. Then set the power voltage to 25 V and adjust the current to 300 mA for 20 min to perform the electrophoresis procedure. Cells were stained with PI. Individual cells were viewed using Olympus IX73 fluorescence microscope.
2.6. Western Blotting. Treated cells were washed with PBS twice and then harvested using ice-cold RIPA lysis buffer containing protease inhibitor PMSF (Gibco) and protein phosphatase inhibitor cocktail (Gibco). The lysates were centrifuged at 12,500 g for 20 min at 4 ∘ C and the supernatant fractions were collected. Protein concentrations were measured with BCA Protein Assay Kit (Gibco). After denaturation at 95 ∘ C for 10 min, equivalent aliquots of protein samples (30 g) were loaded and electrophoresed on SDS-PAGE gels and then transferred to PVDF membrane (Thermo Scientific). The membranes were firstly blocked with 5% nonfat dry milk for 2 h at room temperature and then incubated with primary antibodies (1 : 3000) overnight at 4 ∘ C. Then HRPlinked secondary antibodies (1 : 5000) were incubated for 4 h at room temperature. The bands were visualized with the ChemiDoc6 MP Imaging System (Bio-Rad).

MDC Staining.
MDC staining used to detect the formation of acidic vesicular organelles in Cuc B-treated cells was performed as in our previous reports [28,34].

Measurement of Intracellular ROS.
The effect of Cuc B on ROS formation was determined as in our previous reports [27,38].

Apoptosis
Assay. The apoptosis rates after treatment with Cuc B for 6 h were determined by Annexin V/PI double staining by flow cytometry (BD FACSCanto).

Hoechst 33342
Staining. The condensation of DNA was detected by Hoechst 33342 staining as in our previous report [39].

Statistical
Analysis. Data were expressed as the means ± SEM from at least three separate experiments performed in triplicate. The differences between groups were analyzed using Prism 5.0 (GraphPad Software Inc., San Diego, CA) and < 0.05 is considered statistically significant.

Cuc B-Induced DNA Damage Activated ATM and ATR.
The comet assay showed that significant long tails were observed in Cuc B-treated cells (Figure 2(a)) indicating the occurrence of DNA damage. Cuc B induced -H 2 AX expression in dose- (Figure 2(b)) and time-dependent manner ( Figure 2(c)). Furthermore, the phosphorylation of both ATM/ATR and Chk1/Chk2 was increased in a dosedependent manner (Figures 2(d) and 2(e)). In addition, Cuc B-induced p-Chk1 and p-ATM were downregulated by both KU55933 (Figure 2(f)) and caffeine ( Figure 2(g)).

Cuc B Induced Protective Autophagy.
Compared with the control group, Cuc B-treated cells showed dramatic increase of intensive green fluorescence in MDC staining suggesting the accumulation of autophagic vacuoles (Figure 3(a)). Cuc B treatment led to upregulation of LC3II/LC3I expression in dose- (Figure 3(b)) and time-dependent manner (Figure 3(c)). Furthermore, Cuc B treatment decreased the expressions of p-mTOR, p-AKT, and p62 and increased expressions of p-ULK1 without affecting total AKT and ULK1 (Figure 3(d)). In addition, 3-MA (Figure 3(e)) and CQ (Figure 3(f)) pretreatment further increased Cuc B-induced cell death.

Cuc B Induced
Caspase-Mediated Apoptosis. Cuc B induced apoptosis in a dose-dependent manner (Figure 4(a)). Furthermore, the expression of proapoptotic Bik and Bak was increased while the expression of antiapoptotic protein Bcl-2 was slightly decreased (Figure 4(b)). Increased cleavage of caspase 9, caspase 7, and PARP was also observed (Figure 4(c)). In addition, condensed chromatin was observed in Hoechst 33342 staining after Cuc B treatment (Figure 4(d)).

Cuc B-Induced ROS Formation Resulted in Cell Death.
Cuc B significantly induced ROS formation in a dosedependent manner after 1 h treatment ( Figure 5(a)), which was dramatically decreased at 6 h ( Figure 5 (Figure 6(a)). Furthermore, the deregulated autophagy-related proteins expression such as decreased expression of p-mTOR and p-AKT and increased expression of p-ULK1 and LC3II was also reversed by NAC ( Figure 6(b)). NAC alone also decreased expression of p-AKT. In addition, the cleaved caspases and PARP were reversed by NAC pretreatment (Figure 6(c)).

DNA Damage Mediated Autophagy in Response to Cuc B.
Since both DNA damage and autophagy were initiated by Cuc B, their relationship was clarified by applying DNA damage response inhibitors KU55933 and caffeine and autophagy inhibitors 3-MA and CQ. KU55933 and caffeine reversed Cuc B-induced decrease of p-mTOR and p-AKT (Figures 7(a) and 7(b)). They also reversed Cuc B-induced LC3II, p-ATR, and p-Chk2 (Figure 7(c)). However, 3-MA and CQ showed no effect on -H 2 AX expression (Figures 7(d) and 7(e)). In addition, the long tails caused by Cuc B were not affected by either 3-MA or CQ (Figure 7(f)).

PTEN Bridged DNA Damage and Autophagy in Response to Cuc B.
To dissect the role of PTEN in Cuc B-induced DNA damage, comet assay was performed. PTEN silence showed no effect on the lengths of comet tails (Figure 9(a)) and p-ATM expression but significantly decreased p-Chk1 expression in response to Cuc B (Figure 9(b)), while silence ATM dramatically inhibited Cuc B-induced phosphorylation of both PTEN and Chk1 (Figure 9(c)). Silence Chk1 showed no effect on Cuc B-induced p-PTEN and p-ATM (Figure 9(d)). In addition, silence PTEN reversed Cuc B-induced decrease of p-mTOR and p-AKT and increase of LC3II (Figure 9(e)).

Discussion
We previously reported that Cuc B induced ROS-mediated DNA damage in A549 cells [27]. The main findings of this study include the following: (1) Cuc B induced DNA damage, apoptosis, and protective autophagy in BEL-7402 cells.  PTEN activated by DNA damage bridged DNA damage and autophagy in response to Cuc B. Cuc B-induced cell death was well established in many cell lines. The MTT and colony formation results showed that Cuc B decreased cell viability and inhibited proliferation at nano-mol levels. Increased LDH release suggested that Cuc B might affect cell membrane. We previously reported that Cuc B induced DNA damage in A549 [27], K562 [23], and MCF-7 cells [28]. Cuc B induced long tails in comet assay and increased -H 2 AX expression suggesting that it Oxidative Medicine and Cellular Longevity induced DNA damage in BEL-7402 cells. The key regulators in response to DNA damage are ATM and ATR kinases, which activated Chk1 and Chk2 [40]. The phosphorylation of ATM/ATR and Chk1/Chk2 was increased by Cuc B, which were dramatically inhibited by ATM inhibitor, KU55933 [41], and ATM/ATR inhibitor caffeine [42]. Thus, Cuc Binduced DNA damage response was mediated by ATM/ATR pathways.
Cuc B-induced autophagy was observed in Jurkat [22] and MCF-7 cells [28]. MDC staining for detecting autophagic vacuoles [43] and increased LC3II expression were simple methods for autophagy assay. The AKT/mTOR pathway, especially the mTOR, has been implicated as the central regulator of autophagy in response to natural products [6]. ULK1, a mammalian serine/threonine protein kinase, plays a key role in the initial stages of autophagy by forming a complex with Atg13 and FIP200 to mediate mTOR signaling [44]. Here, Cuc B increased MDC fluorescence, inactivated AKT/mTOR pathway, and upregulated p-ULK1 and LC3II expression, which suggested that Cuc B induced autophagy mediated by AKT/mTOR pathway. Similar results were observed in MCF-7 cells [28]. Autophagy generally acted as a prosurvival role in response to lethal stress. Protective autophagy was reported in Cuc B-treated MCF-7 [28], Cuc Etreated 95D [34], and Cuc I-treated glioblastoma multiforme cells [32]. Cuc B-induced cell death was further enhanced by autophagy inhibitors 3-MA and CQ suggesting that Cuc B induced protective autophagy in BEL-7402 cells.
Induction of apoptosis by Cuc B was documented. Cuc B induced apoptosis in BEL-7402 cells as evidenced by Annexin V/PI double staining and the Hoechst 33342 staining. Furthermore, Cuc B increased the proapoptotic proteins Bak and Bik expression. However, the antiapoptotic protein Bcl-2 was slightly decreased by Cuc B. Thus, Cuc B-induced apoptosis might be mainly through the upregulation of proapoptotic Bcl-2 family proteins. In addition, the increased cleavage of caspase-7, caspase-9, and PARP revealed that apoptosis was caspase-dependent.
Cuc B-induced ROS played important roles in DNA damage, apoptosis, and autophagy [23,26,27,29]. Here, Cuc B-induced ROS formation was also observed in BEL-7402 cells. Furthermore, Cuc B-induced ROS was increased as early as after 1 h treatment suggesting that ROS formation was an early event. NAC dramatically inhibited Cuc Binduced protein expression related to DNA damage, apoptosis, and autophagy. Thus, ROS mediated Cuc B-induced DNA damage, apoptosis, and autophagy in BEL-7402 cells. DNA damage-induced apoptosis has been well recognized while its role in autophagy remains unclear [45]. Here, we found that Cuc B-induced autophagy was inhibited by KU55933 and caffeine while 3-MA and CQ showed no effect on DNA damage. Collectively, the present data suggested that DNA response triggered autophagy in response to Cuc B. It is interesting to note that p-AKT was decreased by NAC treatment. Similar result was reported in oral cancer cells [46]. We considered that Cuc B-induced massive DNA damage stress led to AKT depression while NAC reversed this depression by inhibiting DNA damage through scavenging ROS.
PTEN, a tumor suppressor gene, has been demonstrated to play a critical role in DNA damage repair and DNA damage response [47]. It also opposes PI3K function, negatively regulates PI3K/AKT pathway, and thus leads to inactivation of AKT and mTOR signaling [48]. A recent study showed that Cuc B inhibited SH-SY5Y cells proliferation through upregulation of PTEN [49]. Here, we found that Cuc B increased p-PTEN expression in BEL-7402 cells, which was inhibited by DNA damage inhibitors and NAC suggesting that activation of PTEN was mediated by DNA damage following ROS generation. Silence PTEN showed no effect in comet assay suggesting that PTEN was not involved in Cuc B-induced DNA damage although decreased Chk1 was also observed. Silence ATM decreased Cuc B-induced PTEN expression while silence PTEN did not affect ATM expression, suggesting that ATM activation resulted in PTEN upregulation. Furthermore, silence PTEN reversed Cuc B-induced autophagy-related protein expression suggesting that PTEN was involved in Cuc B-induced protective autophagy. This was further supported by the enhanced cytotoxicity of Cuc B in PTEN silenced cells. These results were consistent with a recent report showing that ATM mediated PTEN phosphorylation and autophagy in response to DNA-damaging agents in A549 cells [36]. Collectively, these results showed that PTEN activation by DNA damage might act as an upstream molecule of autophagy.
In summary, as depicted in Figure 10, this study showed that a natural product, Cuc B, induced ROS-mediated DNA damage, apoptosis, and protective autophagy. The DNA damage activated PTEN linked the crosstalk between DNA damage and autophagy. This study provides potential roles of PETN in the interplay of prodeath DNA damage and the prosurvival autophagy.