Induction of endoplasmic reticulum stress and mitochondrial dysfunction dependent apoptosis signaling pathway in human renal cancer cells by norcantharidin

Previous studies reported that norcantharidin (NCTD) has anti-tumor effects. We investigated the antitumor effects and underlying mechanism of NCTD on human renal cancer in vitro and in vivo. NCTD significantly decreased renal cancer cell viability by induction of apoptosis, as determined by the MTT assay and annexin V/PI staining. NCTD treatment of 786-O and A-498 cells altered the expression of caspase family proteins and PARP. Moreover, NCTD induced mitochondrial depolarization, which was accompanied by an increased level of Bax and decreased levels of Bcl-2 and Mcl-1. NCTD induced endoplasmic reticulum (ER) stress by increasing the expression of Grp78, p-elF2α, ATF4, and CHOP. Pretreatment with an ER stress inhibitor (salubrinal) significantly attenuated the effect of NCTD. NCTD also induced activation of the AKT pathway in 786-O and A-498 cells. Overexpression of AKT partly reversed the effect of NCTD on apoptosis. NCTD treatment led to decreased expression of Bcl-2 and Mcl-1, and increased expression of Bax, cleaved-caspase-9, cleaved-PARP, and p-elF2α. Our in vivo studies demonstrated that NCTD significantly inhibited tumor growth in a nude mouse xenograft model. Taken together, our results suggest that NCTD is a potential anti-tumor agent for treatment of renal carcinoma.


INTRODUCTION
Renal cell carcinoma (RCC) is the ten most frequently occurring human cancers [1]. RCC develops from the proximal renal tubular epithelial cells of the kidneys, and accounts for about 85% of renal cancers [2]. The current treatments consist of chemotherapy and immunotherapy, and are associated with numerous toxicities [3]. Therefore, it is necessary to better understand the molecular mechanisms of this cancer so that new anti-RCC molecular targets can be identified and effective and less toxic drugs can be developed.
Norcantharidin (NCTD, Figure 1A), the demethylated analog of cantharidin, is isolated from natural blister beetles [4]. NCTD has anti-tumor [5], proapoptotic [6,7], anti-metastatic [8], and anti-angiogenic [9] effects. NCTD also has diverse anticancer activities against various types of tumor cells, such as prostate cancer, lung cancer, breast cancer, and colorectal carcinoma [10][11][12][13]. NCTD induces apoptosis through regulation of cell cycle-related proteins, leading to cell cycle arrest at the G2/M phase [12,14]. Other studies reported that NCTD induces mitochondria-dependent apoptosis through inhibition of the AKT/FOXO4/Mcl-1 signaling pathway in human prostate cancer [15] and induces production of reactive oxygen species (ROS) via upregulation of the p38 MAPK pathway in human urinary bladder carcinoma cells [16]. Furthermore, a combination treatment consisting of NCTD with ABT-737 has increased therapeutic efficacy against HCC cells [17]. Nevertheless, the underlying molecular mechanisms of the anti-tumor effects of NCTD on renal cancer cells are still unknown.
Previous studies demonstrated that an imbalance of proteins in the Bcl-2 family, which have roles in the intrinsic apoptosis pathway, leads to increased mitochondrial permeability, release of cytochrome c, and increased caspase-9/caspase-3 expression [18]. There is also evidence that induction of endoplasmic reticulum (ER) stress proteins has a role in apoptosis in various cancer cells [19,20]. The UPR involves three proteins, including inositol-requiring enzyme-1 (IRE1), activating transcription factor 6 (ATF6) and PKR-like ER kinase (PERK)-respond to the accumulation of unfolded proteins as part of a survival response [21]. When the intracellular misfolded or unfolded protein accumulation, ER releases a large number of Bip protein to help the accumulation of protein folding. The accumulation of unfolded protein is reduced, the ER function is restored [22]. So far, no studies have yet examined the effect of NCTD on induction of ER-stress induced apoptosis of tumor cells. The present study examines the anticancer activity of NCTD against renal cancer cells in vitro and in vivo.

NCTD reduces viability of human renal cancer cells
We initially investigated the effect of NCTD on the viability of four human renal cancer cells (786-O, A-498, CaKi-1, and ACHN) and normal proximal tubule epithelial cells (HK-2) using the MTT assay. NCTD reduced the viability of each of the four cell lines of renal cancer cells in a time-and concentration-dependent manner ( Figure 1B-1E). However, the same concentrations and treatment durations only had a mild cytotoxic effect in the normal proximal tubule epithelial (HK-2) cells ( Figure 1F). The IC50 values in the renal cancer cells and proximal tubule epithelial cells were 62.4 ± 5.7 μM (CaKi-1), 43  88.3 ± 3.8 μM (HK-2) after 48 h treatment, respectively ( Figure 1B-1F). These data suggest that NCTD selectively inhibits renal cancer cells at concentrations that are not toxic to normal proximal tubule epithelial (HK-2) cells.

NCTD induces apoptosis and alters expression of apoptosis-associated proteins in human renal cancer cells
We next determined the effect of NCTD on induction of apoptosis in 786-O and A-498 cells by use of flow cytometry with propidium iodide (PI) staining ( Figure 2A). The results indicate that NCTD led to an accumulation of cells in sub-G1 phase after 24 h in a concentrationdependent manner. In particular, the percentage of 786-O cells at sub-G1 was 20.3% at 40 µM NCTD and 36.8% at 80 µM NCTD; the percentage of A-498 cells at sub-G1 was 21.5% at 40 µM NCTD and 43.5% at 80 µM NCTD. We also examined the effect of NCTD on 786-O and A-498 using Annexin V/PI staining (an apoptosis assay) with flow cytometry. The results indicate that the number of Annexin V-FITC and PI positive cells increased as NCTD concentration increased ( Figure 2B). We examined the effect of NCTD on the expression of several critical apoptosis-related proteins (caspase-3, caspase-6, caspase-7, caspase-8, caspase-9, and PARP) in 786-O and A-489 cells. NCTD induced accumulation of cleaved PARP and of activated caspases in a concentration-dependent manner ( Figure 2C). To confirm the contribution of caspase activation on NCTD-induced apoptosis, we pretreated 786-O cells with a pan-caspase inhibitor (Z-VAD-FMK) markedly reversed the effect of NCTD on cell viability ( Figure 2D) and apoptosis ( Figure 2E). These in vitro results confirm that NCTD induces apoptosis in renal cancer cells.
The loss of mitochondrial membrane potential can be triggered by the imbalance of Bax/Bcl-2 leading to the activated process of caspase-9. As shown in Figure 3A, NCTD induced a dose-dependent reduction in mitochondrial membrane potential in human renal cancer cells. We also found that NCTD upregulated Bax expression and downregulated Bcl-2 and Mcl-1 expression in a concentration-dependent manner ( Figure 3B). These in vitro results demonstrate that NCTD-induced apoptosis accompanies mitochondrial dysfunction in human renal cancer cells.

NCTD induces endoplasmic reticulum stress in 786-O cells
Several studies have reported the induction of ER stress during the apoptosis of various tumor cells [23]. Thus we determined the effect of NCTD on ER stress using GFP-labeled endoplasmic reticulum as an intracellular probe to assess stress level. The results show that GFP fluorescence increased in a concentration-dependent manner after NCTD treatment for 24 h, indicative of increased ER stress ( Figure 4A). Several previous studies have previously reported that an unfolded protein response (UPR) induces PERK-mediated phosphorylation of eukaryotic initiation factor-2α, and the preferential translation of ATF-4 [23]. Grp78 is required to restore ER function, and ATF-4 also induces the expression of the transcriptional regulator CHOP, leading to induction of apoptosis [24]. Our results indicate that NCTD significantly increased the expression of Grp78, p-eIF2α, ATF-4, and CHOP in a concentrationdependent manner in 786-O cells ( Figure 4B). These in vitro results suggest that NCTD triggers ER stress in human renal cancer cells.

NCTD-induced ER stress leads to apoptosis of 786-O cells
Next, we sought to confirm that NCTD-induced ER stress leads to induction of apoptosis in renal cancer cells. Thus, we examined the effect of salubrinal, an ER stress inhibitor, on the response. The results show that salubrinal partly reversed the effect of NCTD on cell viability and apoptosis ( Figure 5A). In agreement, annexin V/PI double staining indicated that salubrinal partly inhibited NCTDinduced apoptosis ( Figure 5B), and western blotting showed that salubrinal partly reversed the effect of NCTD on expression of p-eIF2α, ATF-4, and CHOP in 786-O cells ( Figure 5C). Overall, these results indicate that NCTD-induced ER stress is responsible for its induction of apoptosis.

NCTD-induced AKT inactivation depends on ER stress-dependent apoptosis
Next, we determined the role of the MAPK and AKT pathways on NCTD-induced apoptosis. Thus, we incubated 786-O and A-498 cells different concentrations of NCTD for 24 h, and performed western blotting analysis of proteins that have established roles in these pathways. The results show that NCTD reduced the activation of AKT in a dose-dependent manner, but did not affect ERK, p38, or JNK activation ( Figure 6). We also investigated the role of ER stress on NCTD-inhibited AKT activation by simultaneous transfection of cells with a constitutive-AKT (HA-AKT) plasmid and treatment with NCTD (40 µM). The results show that the HA-AKT plasmid partly reversed the effect of NCTD on cell viability ( Figure 7A) and apoptosis ( Figure 7B). In addition, the HA-AKT plasmid also partly reversed the effect of NCTD on the expression of Bcl-2, Bax, p-eIF2α, cleaved-caspase-9, cleaved-PARP, and Mcl-1 ( Figure 7C). These results demonstrate that AKT inactivation plays an essential role in NCTD-mediated apoptosis in human renal cancer cells.

Antitumor effect of NCTD in vivo
Finally, we investigated the effect of NCTD on tumor growth in vivo using 786-O xenograft nude www.impactjournals.com/oncotarget BALC/c mice ( Figure 8A). After 28 days, tumor volume ( Figure 8B) and weight ( Figure 8C) were significantly lower in mice treated with NCTD at doses of 10 and 20 mg/kg, concentrations that did not significantly alter total body weight ( Figure 8D). Immunohistochemistry indicated that Ki-67 was strongly inhibited at NCTD doses of 10 and 20 mg/kg ( Figure 8E). These results indicate that NCTD reduced tumor growth in vivo at levels that had no apparent toxic effects.

DISCUSSION
Additional anticancer drugs are needed to improve the outcome of patients with renal cancer. Many previous studies have reported that NCTD is a safe and effective treatment for many types of tumors [10], but little is known about its effect on renal cancer. Our results indicate that NCTD suppresses the growth of human renal cancer cells in vitro and in vivo by induction of cell cycle arrest and apoptosis, and this is due to its effect on ER stress and the AKT signaling pathway.
Mitochondria are important mediators of the intrinsic apoptosis pathway [25]. At the onset of apoptosis, there are changes in the outer mitochondrial membrane; in particular, changes in Bcl-2 family proteins alter the mitochondrial membrane potential [26]. This protein family has anti-apoptotic proteins, such as Bcl-xL and Bcl-2, and pro-apoptotic proteins, such as Bax [27]. Previous research on gastric cancer cells indicated that NCTD induced mitochondria-dependent cell apoptosis through activation of Bax and the release of cytochrome c, AIF, and Endo G into the cytosol [28]. In addition, Liu et al. found that NCTD increased apoptosis by alteration  The protein expression level of Grp78, ATF-4, CHOP, p-eIF2α and eIF2α were assessed by western blotting, β-actin used as an internal control. All data are represented as mean ± SEM (n = 3) for each group. ** p < 0.01 compared with control. www.impactjournals.com/oncotarget of TR3 and Bcl-2 in melanoma cells in vitro and in vivo [7]. Our experiments with 786-O cells indicate that NCTD treatment led to loss of the MMP, and that this was accompanied by an increase of the pro-apoptotic protein Bax, and decreases of the anti-apoptotic proteins Bcl-2 and Mcl-1.
The ER stress response occurs in diverse biological systems, and has roles in the regulation of cell proliferation, apoptosis, and autophagy in different types of tumor cells [23,29]. Grp78 is a major regulator of ER stress. In particular, this protein functions as a molecular chaperone that maintains the integrity of the ER, and  controls activation of UPR signaling molecules [30]. PERK dissociates from Grp78/BiP, and activates itself by oligomerization and phosphorylation, which directly phosphorylates the translation initiation factor eIF2α, leading to a general attenuation of protein synthesis [31]. However, activation of PERK also leads to increased expression of ATF4 and its targeted transcription factor CHOP (C/EBP homologous protein) during ER stress [32]. Our studies of 786-O cells demonstrated that NCTD-induced apoptosis correlated with ER stress and expression of ER stress-related proteins, such as p-eIF2α, CHOP, and ATF4 ( Figure 4B). What is the relationship between NCTD-induced ER stress and apoptosis? NCTD induces the activation of Grp78 during the early stages of apoptosis, and then activates the transcription factor ATF4 and binding to the CHOP promoter. After this, there is an increase of mitochondrial membrane permeability due to dephosphorylation of AKT. Thus ATF-4/CHOP appears to mediate the apoptotic signals from the ER to the mitochondria. Another possibility is that anticancer drugs induce an excess of ROS, leading to increased apoptosis, possibly through activation of the ER stressmediated apoptotic pathway [33,34]. Inhibition of ER stress by salubrinal attenuated NCTD-induced apoptosis and the effect of NCTD on expression of eIF2a/ATF-4/ CHOP. These results indicate that NCTD induced ER stress, and this led to increased apoptosis mediated by Grp78-phospho-eIF2α-ATF4-CHOP in 786-O cells.
PI3K/AKT activation plays an important role in the ER-stress-mediated regulation of different cell responses, such as proliferation, apoptosis, differentiation, and senescence [35,36]. Recent studies found that inactivation of AKT is also required for the induction of ER stressmediated apoptosis by certain phytochemicals, such as flavokawain C and cirsimaritin, and this is accompanied by inactivation of AKT [37,38]. The phytochemical wogonin has potent cytotoxic effects, in that it induces ROS production and UPR activation through inhibition of AKT activation, leading to increased apoptosis of hepatocellular carcinoma cells [39] and HL-60 leukemia cells [40]. We found that NCTD inhibited the activation of AKT in 786-O and A-498 cells. Furthermore, overexpression of AKT attenuated the NCTD-induced apoptosis and ER stress in 786-O cells. These results suggest that NCTD induces apoptosis via the ER-dependent AKT apoptotic pathway.

Cell viability assay
Cell growth was determined using the MTT assay, as previously described [41]. After treatment with NCTD for 24 h, 1 mL of the MTT reagent (0.5 mg/ml) was added to each well of a multi-well culture plate, and the plate was then incubated for 4 h at 37°C. The absorbance was

Measurement of ER stress
After respective treatment cells were seeded on 8 well Lab-Tek Chambered coverglass (Thermo, Rochester, NY) and fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X100, stained with Hoechst 33342 reagent. The ER-ID Red Assay Kit (Enzo Life Sciences, Lörrach, Germany) was used for staining of the ER according to the manufacturer's instructions. Samples were mounted and photographed under an immunofluorescence microscopy. Quantify the fluorescence density of NCTD treated cells with ER-ID Red Assay Kit by FACSCalibur flow cytometer and the data were analyzed by Cell Quest software (BD Bioscience, Bedford, MA).

Transfection assay
Cells seeded in 6-cm culture dish were transfected at 80% confluency with 3 µg HA-AKT plasmids using TurboFect transfection reagent (Thermo Fisher Scientific Inc.) according to the manufacturer's instructions. Twentyfour hours following transfection, cells were treated with NCTD (40 µM) and harvested after 24 h. AKT overexpression were monitored and verified by western blotting analysis.

Western blot analysis
Western blotting analyses were performed as described previously [42]. Equal amounts of protein extracts (25 μg) were subjected to 10% or 12% SDS-PAGE, and then blotted onto PVDF membranes. The membranes were blocked for 1 h at room temperature using 5% nonfat milk in PBST, and then incubated with the primary antibodies in TBST overnight at 4°C. Proteins were then detected by enhanced chemiluminescence using the Immobilon Western-HRP Substrate (Millipore, Billerica, MA, USA).

Xenograft mouse model
A xenograft mouse model was used to evaluate the effect of NCTD on tumor growth in vivo, as previous described [15]. Each BALB/c male mouse received a subcutaneous inoculation of 786-O cells (5 × 10 6 /0.1 mL) for 7 days. Mice were divided into three groups, with five animals per group. These mice received 0, 10, or 20 mg/kg body weight of NCTD by oral gavage twice per week. When the tumors of control group reached around 800 mm 3 , all mice were killed and the tumors were excised and weighed.

Immunohistochemistry
Immunohistochemical studies were performed as previously described [43]. Tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned at a thickness of 4 µm. These sections were then deparaffinized, hydrated, and immersed three times in PBS. Then, the antigen was retrieved by pre-treatment in a microwave oven for 10 min with 10 mM citrate buffer (pH 6.0). The sections were incubated in 3% H 2 O 2 for 10 min at room temperature to eliminate the activity of endogenous peroxidase. After washing in PBS, the sections were blocked in 5% normal goat serum for 15 min at room temperature. Slides were drained and incubated with the primary antibody anti-Ki-67 (1:200) at 4°C overnight. Then, the slides were incubated in the secondary antibody at 37°C for 15 min, and peroxidase activity was visualized by adding a standard diaminobenzidine/hydrogen peroxide solution for 2 min. The sections were counterstained with hematoxylin, and observed under a light microscope (Nikon, Japan).

Statistical analysis
All experiments were repeated at least three times independently. Data are expressed as means ± standard deviations and analyzed with SPSS version 10.0 (SPSS, Inc., Chicago, IL, USA) and GraphPad PRISM (version 6.0; Graph Pad Software) by Student's t-test or analysis of variance (ANOVA). A p value of 0.05 or 0.01 was considered significant.

CONFLICTS OF INTEREST
None.