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Introduction

Breast cancer is one of the most common cancers in women, and triple-negative breast cancer (TNBC) is the most aggressive breast cancer, resulting in a poor outcome because of high molecular heterogeneity and metastatic potential, and a lack of therapeutic targets (Chang-Qing et al., ). TNBCs do not express human epidermal growth factor receptor 2 (HER2), estrogen receptor alpha (ERα), or progesterone receptor (PR) (Venema et al., ). Due to the absence of these receptors, TNBC does not respond to endocrine therapies, such as tamoxifen, and to HER2-targeting therapies such as herceptin (Jiang et al., ). Chemotherapy is still one of the main approaches to treat TNBC. Traditional first-line drugs for treating breast cancer, such as paclitaxel, are not very effective against TNBC. Second-line drugs that are used to treat breast cancer, such as cisplatin, appear to be more effective against TNBC. However, approximately one-third of patients relapse within 3 years of an adjuvant therapy. Therefore, patients with TNBC have a poor prognosis compared with patients with other types of breast cancer (Zhao et al., ).

Protein kinase RNA-like endoplasmic reticulum kinase (PERK) plays an important role in the unfolded protein response (UPR) elicited by endoplasmic reticulum (ER) stress (Han et al., ; Liu J. et al., ). ER stress, due to misfolded protein accumulation in the ER, activates PERK, which subsequently phosphorylates eukaryotic translation initiation factor 2 alpha (eIF2α) and transiently inhibits global protein translation, while selectively increasing the expression of activating transcription factor 4 (ATF4). Subsequently, ATF4 triggers the expression of some genes, including CCAAT-enhancer binding protein (C/EBP) homologous protein transcription factor (CHOP), and restores ER homeostasis (Cubillos-Ruiz et al., ; Wang et al., ; Dadey et al., ). The activated PERK pathway directly phosphorylates both nuclear factor (erythroid-derived 2)-like-2 (Nrf2) and forkhead box protein O1 (FOXO) proteins, triggering antioxidant and cell survival mechanisms (He et al., ). Additionally, PERK activates AKT signaling to promote cell survival under ER stress conditions (Siwecka et al., ). However, under prolonged ER stress, PERK promotes ER stress-induced cell death via ATF4/CHOP signaling (Kilberg et al., ; Siwecka et al., ).

CCT is a selective eIF2α/PERK activator with potent antiproliferative activity at low millimolar concentrations against human colon cancer cells and chemo-sensitizing activity in U-2 OS human osteosarcoma cells (Stockwell et al., ). CCT was found to ameliorate progressive supranuclear palsy by increasing the level of phosphorylated PERK and Nrf2 (Bruch et al., ). However, the pharmacological effects of CCT have not been comprehensively studied. Hence, we aimed to explore the effects of CCT on TNBC and elucidate its mechanism of action.

Materials and Methods

Reagents

CCT was purchased from MedChemExpress (Monmouth Junction, NJ, USA). Female nude mice (aged 5 weeks, weighing 18&#x;22 g) were procured from Beijing HFK Bioscience Co., Ltd. (Beijing, China). Matrigel was purchased from BD Biosciences (Franklin Lakes, NJ, USA). The detailed information of primary antibodies against cyclin-dependent kinase 4 (CDK4), CDK6, cyclin D1, B-cell lymphoma 2 (Bcl-2), Bclassociated X protein (Bax), cleaved poly (ADP-ribose) polymerase (PARP), PERK, phosphorylated PERK (p-PERK), eIF2α, p-eIF2α, ATF4, CHOP, protein kinase B (AKT), p-AKT, mammalian target of rapamycin (mTOR), p-mTOR, and Ki is presented in Supplementary Table S1. Goat anti-rabbit immunoglobulin G (IgG) and goat anti-mouse IgG secondary antibody were purchased from Cell Signaling Technology (Danvers, MA, USA). SuperSignal West Femto Trial Kit was purchased from Thermo Fisher Scientific (MA, USA). Cell Counting Kit-8 (CCK-8) was purchased from Bimake (Houston, TX, USA). Enhanced BCA Protein Assay Kit, Cell lysis buffer for western blotting and IP, and Annexin V/fluorescein isothiocyanate (FITC) apoptosis detection kit were purchased from Beyotime (Shanghai, China). CHOP RNA interference (RNAi) plasmid was purchased from GenePharma Co., Ltd. (Shanghai, China).

Cell Culture

Human TNBC cell lines, MDA-MB and CAL, were purchased from the American Type Culture Collection (ATCC). The two cell lines were cultured in Leibovitz&#x;s L and RPMI medium supplemented with 10% fetal bovine serum, respectively. The cells were grown at 37°C in a humidified 5% CO₂ atmosphere.

CCK-8 Assay

MDA-MB (8 × 10³ cells/well) and CAL cells (4 × 10³ cells/well) were seeded in well plates and treated with CCT at different doses for 24 or 48 h. Then, 10 µl of CCK-8 solution was added to each well and incubated for 1 or 2 h at 37°C. The absorbance of the sample was measured at nm using a full wavelength microplate reader (Thermo scientific, MA, USA). The viability of cells was calculated relative to the viability of untreated cells.

Colony Formation Assay

CAL cells were seeded in six-well plates ( cells/well) and treated with 0, 4, 6, and 8 μM CCT for 12 days. The colonies were washed three times with cold phosphate-buffered saline (PBS) and fixed in 4% paraformaldehyde for 30 min at room temperature. The surviving colonies were stained with crystal violet for 15 min. The colonies with more than 50 cells were counted under an inverted microscope (Nikon, Tokyo, Japan).

Real-Time Cell Analysis Using xCELLigence

Cell growth was detected in real time using a well-described system (xCELLigence, Roche, Basel, Switzerland). All xCELLigence plates were seeded with MDA-MB (2 × 10⁴ cells/well) and CAL cells (2 × 10³ cells/well) and treated with CCT at different doses on the following day. Cell growth is reported as cell index (CI), which reflects a consistent, logarithmic relationship with the cell number. All experiments were performed in triplicate, and data are presented as mean CI ± standard deviation (SD) over time.

Western Blotting

Total cell proteins and tissue proteins were extracted with RIPA lysis buffer, and the protein concentration was measured using the Enhanced BCA Protein Assay Kit. Subsequently, the proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred on to polyvinyl difluoride membranes. The polyvinyl difluoride membranes were blocked with 5% BSA and incubated with primary antibodies (Supplementary Table S1). Immunoblotting was performed as previously described (Li et al., ). Protein bands were visualized using the SuperSignal West Femto Trial Kit and detected using the Tanon Chemiluminescent Imaging System (Shanghai, China).

Immunohistochemistry (IHC)

Tumor specimens were fixed in 4% paraformaldehyde, and then embedded in paraffin and sectioned. The sections were deparaffinized/rehydrated. The sections were then immersed in citrate unmasking solution and heated in a microwave for antigen unmasking. After blocking with 50 µl of goat serum for 30 min at room temperature, the sections were incubated overnight with primary antibodies (Supplementary Table S1) at 4°C, followed by incubation with secondary antibodies. The signal from each section was visualized with 3&#x;-diaminobenzidine reagent and counterstained with hematoxylin. Images were captured using a light microscope.

Apoptosis Assay

MDA-MB and CAL cells were seeded in six-well plates (1 × 10⁶ cells/well). On the following day, the cells were treated with CCT at different concentrations and incubated for 24 h. The cells were then trypsinized, washed with PBS, and stained using the Annexin V/FITC kit, according to the manufacturer&#x;s protocol. Apoptosis was detected by flow cytometry using the FACScan flow cytometer (Beckman Coulter, CA, USA). The cell population was separated into the following three groups: live cells with a low level of fluorescence (Annexin V-negative, propidium iodide (PI)-negative), early apoptotic cells with green fluorescence (Annexin V-positive, PI-negative), and necrotic and advanced-stage apoptotic cells with both red and green fluorescence (Annexin V-positive, PI-positive).

Cell Cycle Analysis

MDA-MB and CAL cells were seeded in six-well plates (1 × 10⁶ cells/well) and treated with CCT at different concentrations on the following day. After 24 h of incubation, the cells were trypsinized, washed with PBS, and fixed in ice-cold 70% ethanol overnight at 4°C. The cells were subsequently washed three times with PBS and incubated with PI and RNase at 37°C for 1 h. Flow cytometry was performed to analyze the cell cycle data according to the manufacturer&#x;s instructions.

CHOP RNAi Plasmid Transfection In Vitro

MDA-MB and CAL cells were seeded in six-well plates (4 × 10⁵ cells/well) and transfected with 2 μg of CHOP RNAi plasmid and control RNAi plasmid using Lipofectamine and Opti-MEM I reduced serum medium for 6 h. After overnight recovery, MDA-MB and CAL cells were treated with 8 or 10 μM CCT for 24 h, and the cells were collected for western blotting.

Gene Expression Profiling Interactive Analysis (GEPIA) Dataset

GEPIA (cromwellpsi.com) is a newly developed interactive web server for analyzing RNA sequencing data of tumor and normal samples using a standard processing pipeline. It provides customizable functions such as tumor/normal differential expression analysis, profiling according to cancer types or pathological stages, patient survival analysis, similar gene detection, correlation analysis, and dimensionality reduction analysis (Tang et al., ). We used the GEPIA dataset to analyze the AKT/mTOR pathway in breast cancer in relation to CHOP or ATF4, both of which are downstream of PERK.

Orthotopic Implantation Model

The experiments were performed in accordance with the National Guidelines for Animal Care and Use and approved by the Animal Care and Use Committee of Chongqing Medical University. An orthotopic implantation model was established in nude mice by implanting MDA-MB cells (5 × 10⁶ cells/50 µl) mixed with Matrigel () in the mammary fat pad. When the tumors grew to approximately 4&#x;5 mm³, the mice bearing the xenografts were randomly divided into two groups (n = 5/group) and were intraperitoneally injected with corn oil or 24 mg/kg CCT Tumor growth was measured at 2-day intervals after injection. Tumor volume (V) was measured using a slide caliper and calculated using the following formula: V (mm³) = × ab², where a and b represent the long diameter and perpendicular short diameter (mm) of the tumor, respectively.

Statistical Analysis

The data were analyzed with Student&#x;s t-test using GraphPad Prism software (GraphPad, Inc., Chicago, IL, USA). All data are expressed as mean ± SD. The criterion for statistical significance was p <

Results

CCT Suppressed TNBC Cell Viability

The chemical structure of CCT was shown in Figure 1A. To examine the effects of CCT on TNBC cells, we treated MDA-MB and CAL cells with the indicated concentrations of CCT and measured cell viability with CCK CCT significantly inhibited cell viability in a dose-dependent manner (Figures 1B, C). Moreover, we used a real-time cell analysis system to dynamically monitor the cell growth curves of MDA-MB and CAL cells after CCT treatment. CCT inhibited the proliferation of both cell lines in a dose- and time-dependent manner (Figures 1D, E). Furthermore, a colony formation assay showed that 4, 6, and 8 μM CCT significantly inhibited the colony formation of CAL cells in a dose-dependent manner (Figure 1F). Thus, the above results demonstrate the in vitro anti-TNBC effects of CCT

Figure 1 CCT suppressed TNBC cell viability in vitro. (A) The chemical structure of CCT (B, C) MDA-MB cells (B) and CAL cells (C) were treated with CCT at different doses for 24 or 48 h. Cell viability was measured with CCK-8 (n = 5). (D, E) MDA-MB cells (D) and CAL cells (E) were treated with CCT at different doses, cell growth curve assays were performed using real-time cell analysis by xCELLigence (n = 3). (F) CAL cells were treated with various of CCT for 12 days, and the surviving colonies were stained with crystal violet. Colonies with more than 50 cells were counted under an inverted microscope (n = 3). Data are presented as mean ± SD, *p < or **p < vs. control.

CCT Induced the Apoptosis of TNBC Cells

To examine the effects of CCT on the apoptosis of TNBC cells, flow cytometry was used to detect the percentage of apoptotic cells after the treatment of MDA-MB and CAL cells with 0, 6, 8, 10, and 12 μM CCT for 24 h. The proportion of apoptotic cells in both cell lines increased in a dose-dependent manner (Figures 2A, B). Additionally, CCT increased the protein levels of cleaved PARP and the pro-apoptosis protein Bax and decreased the protein levels of the anti-apoptosis protein Bcl-2 in MDA-MB and CAL cells (Figures 2C, D).

Figure 2 CCT induced the apoptosis of TNBC cells. (A, B) MDA-MB cells (A) and CAL cells (B) were treated with CCT at different concentrations for 24 h. Flow cytometry was used to detect apoptosis (n = 3). Data are presented as mean ± SD, *p < or **p < vs. control. (C, D) MDA-MB cells (C) and CAL cells (D) were treated with various of CCT for 24 h, or treated with 8 and 10 μM CCT for indicated time, then cells were collected to detect cleaved PARP, Bax, and Bcl-2 level using Western blotting.

CCT Induced G1 Phase Arrest and Modulated Cell-Cycle-Related Proteins in TNBC Cells

PI staining followed by flow cytometry was used to detect cell cycle distribution after the treatment of MDA-MB and CAL cells with 6, 8, and 10 μM CCT As shown in Figure 3A, the percentages of MDA-MB cells in the G1 phase were ± % (0 μM CCT), ± % (6 μM CCT), ± %, (8 μM CCT), and ± % (10 μM CCT), indicating that CCT induced MDA-MB cell-cycle arrest in the G1 phase in a dose-dependent manner. The effects of CCT on CAL cell cycle arrest were similar to those observed in MDA-MB cells (Figure 3B).

Figure 3 CCT induced G1 phase arrest in TNBC cells. (A, B) MDA-MB cells (A) and CAL cells (B) were treated with CCT at different concentrations for 24 h. The flow cytometry was used to analyze the cell cycle data (n = 3). Data are presented as mean ± SD. (C, D) MDA-MB cells (C) and CAL cells (D) were treated with various of CCT for 24 h, or treated with 8 and 10 μM CCT for indicated time, then cells were collected to detect CDK4, CDK6, and cyclin D1 level using Western blotting.

To explore the molecular action mechanism of CCTinduced cell cycle arrest, western blotting was used to detect the levels of CDKs and cyclin D1&#x;proteins known to play important roles in cell cycle progression. The protein levels of CDK4, CDK6, and cyclin D1 in MDA-MB and CAL cells decreased in a dose- and time-dependent manner with CCT treatment (Figures 3C, D).

CCT Activated PERK/eIF2α/ATF4/CHOP Signaling

CCT is a selective activator of eIF2α/PERK signaling (Bruch et al., ). Therefore, we studied the effects of CCT on expression of proteins in the PERK signaling pathway such as eIF2α, ATF4, and CHOP. MDA-MB and CAL cells were treated with CCT at different concentrations. CCT increased the phosphorylation of PERK and eIF2α in a dose- and time-dependent manner; it also increased the ATF4 and CHOP protein levels (Figures 4A, B). These results indicate that CCT triggers the activation of PERK signaling in TNBC cells.

Figure 4 CCT activated PERK/eIF2α/ATF4/CHOP signaling. (A, B) MDA-MB cells (A) and CAL cells (B) were treated with various of CCT for 24 h, or with 8 and 10 μM CCT for indicated time, then cells were collected to detect PERK, p-PERK, eIF2α, p-eIF2α, ATF4, and CHOP level using Western blotting. (C, D) MDA-MB cells (C) and CAL cells (D) were transfected with 2 μg of CHOP RNAi plasmid using Lipofectamine for 6 h. After overnight recovery, MDA-MB and CAL cells were treated with 8 and 10 μM CCT for 24 h, then cells were collected to detect CHOP, cleaved PARP, and Bax level using Western blotting CCT abbreviated as CCT.

CHOP is a major mediator of downstream signaling of PERK, and PERK activation promotes cell death via CHOP signaling. Hence, we explored the role of CHOP in CCTinduced apoptosis. We knocked down CHOP expression in MDA-MB and CAL cells by RNAi plasmid transfection and treated the cells with CCT Treatment of cells with CCT increased the protein levels of CHOP, cleaved PARP, and Bax. However, treatment of CHOP-silenced cells with CCT decreased the protein levels of CHOP, cleaved PARP, and Bax compared with those in CCTtreated unsilenced cells (Figures 4C, D).

CCT Suppressed AKT/mTOR Signaling in TNBC Cell Lines

AKT/mTOR pathway inhibition reduces tumor progression. First, we used the GEPIA dataset to study the AKT/mTOR pathway members in breast cancer in relation to CHOP or ATF4, both of which are downstream of PERK. We observed a negative correlation between the AKT/mTOR pathway members and CHOP and ATF4 in breast cancer (Figures 5A, B). Next, we examined the effects of CCT on the phosphorylation of AKT and mTOR. The exposure of cells to various concentrations of CCT inhibited the phosphorylation of AKT and mTOR in a time-dependent manner (Figures 5C, D). Thus, our results indicate that the inactivation of the AKT/mTOR signaling pathway may be involved in the anti-TNBC effects of CCT

Figure 5 CCT suppressed AKT/mTOR signaling. (A, B) GEPIA dataset (cromwellpsi.com) was used to analyze the AKT/mTOR pathway in breast cancer in relation to ATF4 (A) and CHOP (B). CHOP also known as DDIT3. (C, D) MDA-MB cells (C) and CAL cells (D) were treated with 8 and 10 μM CCT for indicated time, then cells were collected to detect AKT, p-AKT, mTOR, and p-mTOR level using Western blotting.

CCT Inhibited Tumor Growth in an MDA-MB Cell Orthotopic Xenograft Mouse Model

An MDA-MB orthotopic xenograft mouse model was established to assess whether CCT inhibits in vivo tumor growth. We found that 24 mg/kg CCT inhibited tumor growth after 12 days, and these effects became more apparent after 21 days of CCT treatment (p < ) (Figure 6A). There was no significant change in the body weight of CCTtreated and control mice (Figure 6B). IHC revealed that the Ki protein level was significantly reduced in tumor sections from CCTtreated mice compared with that from the control mice (Figure 6C). These results suggest that CCT has anti-TNBC activity.

Figure 6 CCT inhibited tumor growth in an MDA-MB cell orthotopic xenograft mouse model. (A) Tumor volumes were measured every three days and differed at the end of the treatment period (n = 5). Data are presented as mean ± SD, *p < vs. control. (B) Body weight changes in mice during the 21 days of CCT treatment (n = 5). Data are presented as mean ± SD. (C) Representative tumor tissues were sectioned and subjected to immunohistochemistry staining (magnification, × ). (D) Representative tumor tissues from each group were prepared and subjected to Western blotting assay.

To determine whether the activation of the PERK/ eIF2α/ATF4/CHOP signaling pathway and inactivation of the AKT/mTOR signaling pathway were involved in CCTinduced tumor inhibition, we performed western blotting and IHC. IHC showed that CCT treatment significantly increased the p-eIF2α, ATF4, and CHOP levels in the xenograft model (Figure 6C). This was supported by the Western blotting results (Figure 6D and Supplementary Figure S1). In addition, Western blotting showed that the treatment with CCT caused a significant decrease in the protein levels of CDK4, CDK6, p-AKT, and p-mTOR (Figure 6D and Supplementary Figure S1). Thus, CCT inhibited tumor growth via the activation of the PERK/eIF2α/ATF4/CHOP signaling pathway and inactivation of the AKT/mTOR signaling pathway.

Discussion

To the best of our knowledge, this is the first study to show that CCT, a selective activator of eIF2α/PERK, exerts its anti-TNBC effects by inducing G1 phase arrest and apoptosis via the activation of the PERK/eIF2α/ATF4/CHOP pathway and inactivation of the AKT/mTOR pathway. ER stress triggers the UPR in carcinogenesis (Mcgrath et al., ; Liu J. et al., ; Siwecka et al., ). On one hand, ER stress promotes cell survival and induces drug resistance (Rzymski et al., ; Pandey et al., ; Shi et al., ), and on the other hand, long-term ER stress can become pro-apoptotic (Han et al., ; Liu Y. S. et al., ). UPR consists of three major signaling pathways mediated by inositol-requiring enzyme 1 (IRE-1), ATF6, and PERK. The PERK pathway is a significant determinant of cell fate in UPR (Han et al., ; Pakos-Zebrucka et al., ; Cubillos-Ruiz et al., ; Mohamed et al., ). Short-term activation of PERK promotes cell survival via the induction of eIF2α phosphorylation (Atkins et al., ; Krishnamoorthy et al., ). Prolonged activation of PERK triggers death pathways and leads to apoptosis via the PERK/eIF2α/ATF4 axis and the subsequent activation of CHOP, also known as growth arrest and DNA damage-inducible protein (GADD) (Chen et al., a; Siwecka et al., ). Thus, targeting PERK, a key molecule in UPR-signaling, may be a novel approach for treating cancers (Mcgrath et al., ). As CCT is a selective PERK activator, we first investigated whether CCT activated the PERK/eIF2α/ATF4/CHOP pathway in TNBC. As expected, CCT considerably increased the protein levels of p-eIF2α, ATF4, and CHOP in MDA-MB and CAL cells in a time- and dose-dependent manner.

CCT has been reported to ameliorate progressive supranuclear palsy in mice (Bruch et al., ), inhibit the proliferation of human colon cancer cells, and increase chemosensitivity of U-2 OS human osteosarcoma cells (Stockwell et al., ). However, the effects of CCT on breast cancer have not been reported. In this study, we demonstrated the in vitro anti-TNBC effects of CCT The results of the CCK-8 assay, real-time cell analysis, and colony formation assay showed that CCT reduced MDA-MB and CAL cell viability and proliferation, and colony formation in a dose-dependent manner. Furthermore, CCT induced the apoptosis of MDA-MB and CAL cells as evidenced by the increase in the protein levels of cleaved PARP and Bax and the decrease in the levels of Bcl Additionally, the knockdown of CHOP, the main molecule mediating the prolonged activation of PERK-induced apoptosis, attenuated the CCTinduced increase in the protein levels of cleaved PARP and Bax. Taken together, these findings suggest that CCT induced apoptosis by triggering PERK signaling and ultimately, activating CHOP.

The uncontrolled G1/S checkpoint plays an important role in the progression of human cancers by allowing inappropriate proliferation and distorting fate-driven cell cycle exit (Abuhammad et al., ). The CDK and cyclin D1 complexes are important cell cycle regulators. Cyclin D1 binding to CDK4 and CDK6 drive cell cycle entry and G1 phase progression. The inhibition of cyclin D1 and CDK4/6 has been considered a promising strategy for the treatment of cancers including TNBC (Presti and Quaquarini, ; Wang et al., ). The activation of PERK/eIF2α signaling has been shown to block the translation of cyclin D1 and CDKs, resulting in cell cycle arrest in the G1 phase. CCT promotes eIF2α phosphorylation to inhibit cell cycle G1/S phase transit (Brewer and Diehl, ; Stockwell et al., ). Our results show that CCT induced G1 phase arrest and decreased the CDK4, CDK6, and cyclin D1 levels. We also found that persistent activation of PERK/eIF2α signaling by CCT induced cell cycle G1 phase arrest by suppressing the expression of the CDK-cyclin complex.

Targeting the AKT/mTOR pathway has been considered an attractive approach in cancer treatment (Li et al., ; Chen et al., b; Wen et al., ). PERK and the AKT/mTOR pathway exhibit mutual regulation. Although studies have shown that PERK activates the AKT/mTORC1 signaling pathway to promote cell survival (Bobrovnikova-Marjon et al., ; Koga et al., ), there are some studies showing that persistent activation of PERK signaling inhibits AKT/mTOR signaling (Du et al., ; Ohoka et al., ; Appenzeller-Herzog and Hall, ). In this study, we observed a negative correlation between members of the AKT/mTOR pathway and CHOP and ATF4 in breast cancer, both of which are downstream of PERK. CCT significantly suppressed the protein levels of p-AKT and p-mTOR in MDA-MB and CAL cells. Taken together, these findings indicate that CCT persistently activated PERK/ATF4/CHOP signaling and inhibited Akt/mTOR signaling.

For the past two decades, the inoculation of tumor tissue from patients or human cancer cell lines into immunodeficient rodents is the main approach to establish subcutaneous or orthotopic transplantation tumors. Xenograft models are a major preclinical screen in the development of novel cancer therapeutics (Cl and Pj, ). In the present study, an MDA-MB orthotopic xenograft mouse model was established to evaluate the effects of CCT on tumor growth in vivo. CCT suppressed tumor growth and reduced the protein levels of CDK4 and CDK6 in MDA-MB xenograft mice. CCT increased the levels of p-eIF2α, ATF4, and CHOP, and decreased the levels of p-AKT and p-mTOR. Thus, CCT, a selective eIF2α/PERK activator, inhibited TNBC viability and proliferation by activating the PERK/eIF2α/ATF4/CHOP pathway and inactivating the AKT/mTOR pathway in vivo.

In conclusion, our findings demonstrate that CCT exerts anticancer activity by inducing apoptosis and G1 phase arrest in TNBC cells. The mechanism of this anticancer activity of CCT involves the activation of the PERK/eIF2α/ATF4/CHOP pathway and inactivation of the AKT/mTOR pathway. CCT shows potential to be developed as a therapeutic agent for TNBC. Future studies should explore the efficacy and safety of CCT for TNBC treatment.

Data Availability Statement

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.

Ethics Statement

All animal experiments were approved by the Chongqing Medical University Animal Subjects Ethics Sub-committee and conducted in accordance with the Institutional Guidelines and Animal Ordinance of the Department of Health.

Author Contributions

WZ conceived ideas, designed the experiments, performed the data analyses and wrote the manuscript. XL designed the experiments, performed the experiments and data analyses, and wrote the manuscript. XY, DZ, HZ, and LL performed the experiments. WL, XZ, and BC performed the part of the experiments and contributed reagents and materials. All authors read and approved the submitted version.

Funding

This project is supported by grants from National Natural Science Foundation of China (No. and No), the Science and Technology Project of Chongqing Municipal Education Commission (cromwellpsi.com), Basic Research and Frontier Exploration Project of Yuzhong District of Chongqing (), and Subject Talent Training Program of College of Pharmacy of Chongqing Medical University (cromwellpsi.comG).

Conflict of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Supplementary Material

The Supplementary Material for this article can be found online at: cromwellpsi.com#supplementary-material

References

Results

Initial experiments focused on defining potential mechanisms by which GST-MDA-7 promotes cell killing in four independent primary human glioma cell populations: GBM5, GBM6, GBM12, and GBM14 (23). Treatment of GBM5, GBM6, GBM12, and GBM14 cells with GST-MDA-7 was first noted to induce cell killing ~48 h after exposure and caused significant cell killing within 72 h (Fig. 1A; Table 1; Supplementary Fig. S1).⁹ Cell killing was blocked by a pan-caspase inhibitor and an inhibitor of caspase-9 but not by an inhibitor of caspase Cell killing correlated with increased release of cytochrome c into the cytosol of GBM6 cells and with the cleavage of caspase-3 (Fig. 1A, top). In GBM6 cells, GST-MDA-7 treatment caused inactivation of ERK1/2 that correlated with dephosphorylation of BAD S and dephosphorylation and increased expression of BIM (Fig. 1B). In GBM6 cells, GST-MDA-7 treatment caused activation of JNK that was causal in the activation of BAX and the cleavage of BID and caspase-3 (Fig. 1B). However, the decrease in BCL-x_L protein expression caused by GST-MDA-7 treatment was noted to be JNK independent. In all four glioma isolates, inhibition of caspase-8 function (IETD; expression of CRM A or c-FLIP-s) did not alter GST-MDA-7 lethality, whereas inhibition of caspase-9 function (LEHD; expression of dominant-negative caspase-9 or XIAP or BCL-x_L) significantly reduced GST-MDA-7 lethality as judged by cells becoming PI positive or retaining trypan blue dye (Fig. 1C; Supplementary Fig. S2; data not shown).⁹ GST-MDA-7 lethality correlated with caspasedependent cleavage of pro-caspase-3 (Fig. 1C, top inset).

GST-MDA-7 causes a caspasedependent induction of primary human glioma cell death. A, primary human glioma cells (GBM6) were treated 24 h after plating with GST-MDA-7 (30 nmol/L). At the indicated time points after GST-MDA-7 treatment, GBM6 cells were isolated and subjected to SDS-PAGE, and immunoblotting was done to measure the release of cytochrome c into the cytosol, the expression of cleaved caspase-3 and ²-actin, and cell killing by terminal deoxynucleotidyl transferase&#x;mediated dUTP nick end labeling assay and cell killing by Hoechst assay (n = 2). B,top, GBM6 cells were treated with 1 or 30 nmol/L GST-MDA-7/GST 36 h after plating. Cells were isolated 48 h after exposure and ¼g protein was subjected to SDS-PAGE on 12% gels followed by immunoblotting to determine the phosphorylation status of ERK1/2 and JNK and total expression of ERK2 (n = 4). Bottom, GBM6 cells were cultured for 24 h and then treated with the JNK inhibitory peptide based on sequence from JIP-1 (10 ¼mol/L; JNK-IP) 30 min before addition of GST or GST-MDA-7 (30 nmol/L). Cells were isolated 24 h after GST-MDA-7 treatment and cell lysates were split into two portions. In one portion, equal amounts of protein lysate were subjected to immunoprecipitation using the anti-active BAX 6A7 antibody. Immunoprecipitates were subjected to SDS-PAGE and immunoblotted for BAX. In the second portion, equal amounts of protein lysate were subjected to SDS-PAGE and immunoblotted for phosphorylated JNK (P-JNK), BCL-x_L, cleaved caspase-3, BAX, BAD, BAD S, BIM, and ERK2. Data are from a representative experiment (n = 3). C, GBM6 cells were plated and 12 h after plating infected at a multiplicity of infection of 50 to express no gene (CMV), dominant-negative caspase-9 (dncasp9), or treatment with the JNK inhibitory peptide (10 ¼mol/L) as indicated. Twenty-four hours after infection, cells were treated with 30 nmol/L GST or GST-MDA-7, and 72 h after GST-MDA-7 treatment, cells were isolated and cell viability was determined by using Annexin V/PI staining assays in triplicate using a flow cytometer (±SE; n = 5; #, P  , lower amount of cell killing than vehicle-treated cells). Top inset, at 48 h after 30 nmol/L GST-MDA-7 or GST treatment, GBM6 cells expressing dominant-negative caspase-9 or the caspase-8 inhibitor CRM A were isolated and subjected to SDS-PAGE and immunoblotting was done to measure the expression of ERK2 and the levels of cleaved caspase-3 (n = 2). D, SV40 large T protein transformed MEFs, WT, lacking BIM (BIM&#x;/&#x;), lacking BAK (BAK&#x;/&#x;), lacking BAX (BAX&#x;/&#x;), lacking BAK and BAX (BAK&#x;/&#x;BAX&#x;/&#x;), and lacking BID (BID&#x;/&#x;) were plated and 24 h after plating were treated with nmol/L GST-MDA-7 or GST. Seventy-two hours after GST-MDA-7 treatment, cells were isolated and cell viability was determined by trypan blue exclusion assays in triplicate using a hemacytometer. Data are presented as the true percentage increase in cell death above GST-treated cells (±SE; n = 5; #, P  , lower amount of cell killing than WT cells). Inset, MEF cells were plated and 24 h after plating treated with nmol/L GST-MDA-7 (M7) or GST. Twenty-four and 48 h after GST-MDA-7 treatment, cells were isolated, the cytosol was isolated and subjected to SDS-PAGE, and immunoblotting was done to measure the expression of cytochrome c and glyceraldehydephosphate dehydrogenase (GAPDH; n = 3).

To further investigate mechanisms of GST-MDAinduced mitochondrial dysfunction, we used SV40 large T antigen transformed mouse embryonic fibroblasts (MEF) devoid of expression of defined proapoptotic genes. Loss of BIM or BAK function had a modest but significant effect on GST-MDA-7 lethality, whereas loss of BAX and loss of BAX and BAK expression profoundly reduced toxicity (Fig. 1D). In agreement with data in Fig. 1B, genetic manipulation to delete expression of BID also significantly reduced GST-MDA-7 toxicity. Loss of BAX and BAK function or loss of BID function reduced the ability of GST-MDA-7 to promote cytochrome c release into the cytosol (Fig. 1C, top immunoblotting). In agreement with data showing that BAX activation and BID cleavage were JNK dependent, inhibition of JNK with JNK inhibitory peptide treatment suppressed the toxicity of GST-MDA-7 in GBM6 cells from ± % above the GST control value with vehicle treatment to ± % above the GST control value (±SE; n = 3). Collectively, these findings argue that GST-MDA-7 promoted activation of multiple proteins, which act to induce mitochondrial dysfunction, and that activation of the intrinsic mitochondrial pathway represents an important apoptotic mechanism for this cytokine in transformed cells.

In Fig. 1, we noted that BID function, but not caspase-8 function, correlated with GST-MDAinduced lethality. BID is a substrate for both caspase-8 and cathepsin proteases, and in glioma cells, cathepsin enzymes are overexpressed and play a key role in tumor invasion and angiogenesis (29, 34). GST-MDA-7 toxicity was nearly abolished by the loss of cathepsin B expression comparing appropriate matched immortalized rodent fibroblast cells, of a different lineage to those used in Fig. 1, which correlated with a reduction in the GST-MDAinduced release of cytochrome c into the cytosol of these cells (Fig. 2A). Combined inhibition of caspase-9 and cathepsin function was required to suppress GST-MDA-7 lethality in transformed fibroblasts and in GBM6 and GBM12 cells (Table 2). Loss of cathepsin B function suppressed the GST-MDAinduced degradation of BID and caspase-3 in transformed fibroblasts, and inhibition of cathepsin B function suppressed the GST-MDAinduced degradation of BID in GBM6 cells [Fig. 2B, (i) and (ii)]. The cleavage of p43 cathepsin B after GST-MDA-7 treatment was suppressed by inhibition of JNK [Fig. 2B, (ii)]. Collectively, these findings suggest that GST-MDA-7 induces multiple parallel proapoptotic pathways in transformed cells that converge to cause mitochondrial dysfunction: a JNKdependent activation of BAX; a JNKdependent activation of cathepsin B, leading to a cathepsin B&#x;dependent cleavage of BID; and increased activity of BAD and BIM.

Cathepsin B &#x; dependent cleavage of BID plays an important role in GST-MDA-7 toxicity in transformed cells. A, mouse immortalized embryonic fibroblasts (WT; cathepsin B&#x;/&#x;) were cultured for 36 h and then treated with GST or GST-MDA-7 (0&#x; nmol/L, as indicated). Cells were isolated for viability analyses 72 h after GST-MDA-7 treatment as judged in triplicate by trypan blue dye exclusion assay (n = 3). Top flow cytometry, WT and cathepsin B&#x;/&#x; cells were cultured for 36 h and then treated with GST or GST-MDA-7 ( nmol/L). Cells were isolated for Annexin V/PI viability analyses 72 h after GST-MDA-7 treatment as judged in triplicate (n = 3). Top immunoblotting, 48 hours after GST-MDA-7 treatment, WT and cathepsin B&#x;/&#x; cells were isolated, the cytosol fraction was isolated and subjected to SDS-PAGE, and immunoblotting was done to measure the levels of cytochrome c and GAPDH (n = 3). B,top (i ), MEF cells were isolated for immunoblotting 48 h after GST-MDA-7 treatment, as per A, and ¼g protein at each time point was subjected to SDS-PAGE on 12% gels followed by immunoblotting to determine the total expression of ERK2, pro-caspase-3, and BID (representative blots; n = 3); Bottom (ii ), left, GBM6 cells were cultured for 24 h after plating and then treated with the cathepsin B inhibitor (1 ¼mol/L; cath. B inhib.) 30 min before addition of GST or GST-MDA-7 (30 nmol/L). Cells were isolated 48 h after GST-MDA-7 treatment and ¼g protein at each time point was subjected to SDS-PAGE on 12% gels followed by immunoblotting to determine the total expression of ERK2, pro-caspase-3, and BID (representative blots; n = 3). Right, GBM6 cells were plated and cultured for 36 h and then treated with the JNK inhibitory peptide (10 ¼mol/L) 30 min before addition of GST or GST-MDA-7 (30 nmol/L). Cells were isolated 24 h after GST-MDA-7 treatment and equal amounts of protein lysate were subjected to SDS-PAGE and immunoblotted for expression of p43 cathepsin B and ERK2. Data are from a representative experiment (n = 3).

MDA-7/IL has been suggested to promote ER stress signaling and cell death by binding to and inactivating the PERK-binding protein BiP/GRP78 (19). Treatment of transformed fibroblasts that lacked expression of PERK (PERK&#x;/&#x;) with GST-MDA-7 caused significantly less cell killing than observed in their isogenic matched WT counterparts (Fig. 3A). This correlated with reduced release of cytochrome c into the cytosol of GST-MDA-7 treated PERK&#x;/&#x; cells; cytochrome c release into the cytosol was JNK dependent (Fig. 3B, top blotting). Of note, this observation was the opposite to treating these cells with an established inducer of ER stress, thapsigargin (Supplementary Fig. S3).⁹ Surprisingly, based on known downstream targets of PERK signaling, expression of a dominant-negative eIF2± S51A protein only modestly modified the survival response of GBM6 cells treated with GST-MDA-7 (Supplementary Fig. S4).⁹ GST-MDAinduced BID cleavage, cathepsin B cleavage, suppression of BCL-x_L expression, inhibition of ERK1/2 phosphorylation, and increased eIF2± S51 phosphorylation in transformed fibroblasts were PERK dependent (Fig. 3C).

GST-MDA-7 promotes transformed cell killing through a PERK-dependent pathway. A, transformed MEFs: WT or lacking expression of PERK (PERK&#x;/&#x;) were cultured for 36 h and then treated with GST or GST-MDA-7 (0&#x;60 nmol/L, as indicated). Cells were isolated for viability analyses 72 h after GST-MDA-7 treatment as judged in triplicate by trypan blue dye exclusion assay (±SE; n = 3; *, P  , greater than WT cells). B, WT and PERK&#x;/&#x; cells were cultured for 36 h and then treated with GST or GST-MDA-7 ( nmol/L). Cells were isolated for Annexin V/PI viability analyses 72 h after GST-MDA-7 treatment as judged in triplicate (n = 3). Top immunoblotting, 48 h after GST-MDA-7 and JNK inhibitory peptide treatment, WT and PERK B&#x;/&#x; cells were isolated, the cytosol was isolated and subjected to SDS-PAGE, and immunoblotting was done to measure the expression of cytochrome c and GAPDH (n = 3). C,top (i ), MEFs as indicated (WT, cathepsin B&#x;/&#x;, and PERK&#x;/&#x;) were treated with GST or GST-MDA-7 (60 nmol/L) and 48 h after treatment isolated. Equal amounts of protein lysate were subjected to SDS-PAGE and immunoblotted for expression of p43 cathepsin B, BID, and ERK2 and for phosphorylation of JNK1/2 (P-JNK1/2). Data are from a representative experiment (n = 3). Bottom (ii ), WT and PERK&#x;/&#x; fibroblasts were treated with GST or GST-MDA-7 (60 nmol/L) and 48 h after treatment isolated. Equal amounts of protein lysate were subjected to SDS-PAGE and immunoblotted for expression of BCL-x_L, ERK2, and eIF2± and for phosphorylation of ERK1/2 and eIF2± (S51 ). Data are from a representative experiment (n = 2). D, GBM6 cells were plated and 12 h after plating portions of these cells were transfected with a scrambled siRNA (SCR), a siRNA to knockdown caspase-2 expression (si-2), or a siRNA to knockdown caspase-4 expression (si-4). Other portions of cells were not transfected but treated with either vehicle (DMSO; VEH) or with the JNK inhibitory peptide (10 ¼mol/L). Forty-eight hours after transfection, cells were treated with GST or GST-MDA-7 at the indicated concentrations. Forty-eight hours (immunoblotting inset to the right) or, for viability analyses, 72 h after GST-MDA-7 treatment, cells were isolated as judged in triplicate by trypan blue dye exclusion assay (±SE; n = 2; #, P  , less than scrambled siRNA &#x; transfected or vehicle-treated cells treated with the same concentration of GST-MDA-7).

Considering published reports indicating that ER stress signaling is linked to the activation of the JNK pathway, and our present studies showing that GST-MDAinduced toxicity is JNK dependent and that inhibition of JNK signaling block cytochrome c release, we determined whether loss of PERK expression altered the activation of JNK following GST-MDA-7 exposure (35&#x;38). Treatment of WT-transformed fibroblasts with GST-MDA-7 promoted JNK1/2 activation, predominantly JNK1, which was causal in cell killing (Fig. 3C). In PERK&#x;/&#x; cells, JNK1/2 was very weakly activated by GST-MDA-7, whereas, of note, loss of cathepsin B function did not alter JNK1/2 activation. Hence, GST-MDA-7 induces a PERK-dependent form of ER stress that promotes JNK pathway-dependent activation of BAX and mitochondrial dysfunction as well as promoting a JNK-dependent activation of cathepsin B that acts to cleave and activate BID, thereby likely promoting further BAX activation and mitochondrial dysfunction.

ER stress&#x;induced cell killing can also be mediated by caspase-2 and caspase-4 that can cause mitochondrial dysfunction as well as initiate cell killing directly (39). Knockdown via small interfering RNA (siRNA) of caspase-2 or caspase-4 expression in GBM6 glioma cells partially, albeit significantly, reduced GST-MDA-7 toxicity in this cell line (Fig. 3D; see also inset on top right, confirming siRNA knockdown); GST-MDA-7 caused pro-caspase-2 and pro-caspase-4 cleavage that was JNK dependent (Fig. 3D, bottom right).

Because GST-MDA-7 promotes ER stress signaling, we investigated whether cell killing was associated with lysosomal vacuolization and whether any processes known to be associated with autophagy occurred. Treatment of transformed MEFs with GST-MDA-7 caused vacuolization of acidic compartments within 12 h, as judged by Lyso-tracker Red staining, an effect that was not observed in PERK&#x;/&#x; cells at either 12 or 24 h after GST-MDA-7 treatment [Fig. 4A, (i); data not shown]. As noted previously, 24 h after GST-MDA-7 exposure, relatively little cell induction of cell killing was observed (data not shown). In MEFs expressing a bona fide dominant-negative form of one downstream substrate of PERK, eIF2± S51A, vacuolization of acidic compartments was observed at 12 and 24 h, albeit to a lesser extent than that noted in WT cells. The acidic compartment vacuolization effect in transformed MEFs, and in GBM6 and U cells, was suppressed by a nonspecific inhibitor of autophagy, 3MA, suggestive that cells may be undergoing autophagy [Fig. 4A, (ii)]. GST-MDAinduced vacuolization of acidic compartments was not blocked by inhibition of JNK or p38 mitogen-activated protein kinase (data not shown).

GST-MDA-7 causes vacuolization in transformed fibroblasts in a PERK-dependent and eIF2±-independent manner. A, (i ), left, transformed MEFs (WT; deleted for PERK, PERK&#x;/&#x;, expressing dominant-negative eIF2± S51A, eIF2± S51A) were plated in four-well glass chamber slides in triplicate and 24 h after plating treated with GST or GST-MDA-7 ( nmol/L). Twenty-four hours after GST-MDA-7 treatment, Lysotracker Red was added to the culture medium and the cells were examined under visual light (visible) or under fluorescent light (Lysotracker Red) at ×40 magnification. Representative images from the triplicate plating (n = 2). (ii ), right, WT MEF and U and GBM6 human glioma cells were treated with GST or GST-MDA-7 ( nmol/L) and coincubated for the 24 h GST-MDA-7 treatment with vehicle (PBS) or with 3MA (5 mmol/L). Twenty-four hours after GST-MDA-7 treatment, Lysotracker Red was added to the culture medium and the cells were examined under visual light (visible) or under fluorescent light (Lysotracker Red) at ×40 magnification. Representative images from the triplicate plating (n = 2). B, U cells were plated in four-well chamber glass slides in triplicate and 12 h after plating were transfected with a plasmid to express LC3-GFP. Twelve hours after transfection, cells were pretreated with vehicle (PBS) or with 3MA (5 mmol/L) and 30 min later were treated with GST or GST-MDA-7 ( nmol/L). Twenty-four hours after GST-MDA-7 exposure, the U cells were examined under visual light (visible) or under fluorescent light (LC3-GFP) at ×40 magnification. Representative images from the triplicate plating (n = 2). C, (i ), GBM6 and U cells were plated in four-well chamber glass slides in triplicate and 12 h after plating transfected with a plasmid to express LC3-GFP and in parallel cotransfected with either a vector control plasmid (CMV) or with a plasmid to express dnPERK. Twelve hours after transfection, cells were treated with GST or GST-MDA-7 ( nmol/L). Twenty-four hours after GST-MDA-7 exposure, the GBM6 and U cells were examined under visual light (visible) or under fluorescent light (LC3-GFP). Representative images from the triplicate plating (n = 2). (ii ), U cells were plated in four-well chamber glass slides in triplicate and 12 h after plating transfected with a plasmid to express LC3-GFP and in parallel cotransfected with either a vector control plasmid to express a nonspecific scrambled siRNA (siSCR) or plasmids to knockdown expression of Beclin-1 (siBeclin-1) or ATG5 (siATG5). Parallel studies also transfected cells with plasmids to express scrambled siRNA and untagged GFP. Twelve hours after transfection, cells were treated with GST or GST-MDA-7 ( nmol/L). Twenty-four hours after GST-MDA-7 exposure, the U cells were examined under fluorescent light (LC3-GFP and GFP). Representative images from the triplicate plating (n = 3). Immunoblotting, cells transfected with siRNA constructs to modulate the expression of ATG5 and Beclin-1 were immunoblotted to determine the expression of Beclin-1 and ATG5 48 h after transfection. Cells treated with GST-MDA-7 and GST were immunoblotted 48 h after treatment to determine the expression of Beclin-1, ATG5, the cleavage status of LC3 and GAPDH (n = 2). D, transformed MEFs (WT; deleted for PERK, PERK&#x;/&#x;) 24 h after plating were treated with GST or GST-MDA-7 ( nmol/L). Twenty-four hours after GST-MDA-7 treatment, cells were isolated and subjected to SDS-PAGE to determine the expression of Beclin-1, ATG5, the cleavage status of LC3 and GAPDH (n = 2).

Based on findings showing 3MA-dependent GST-MDAinduced vacuolization of acidic compartments, we determined whether the vacuoles also contain a marker for autophagy, LC3. Cells were transfected with a GFP-tagged form of LC3, treated with GST-MDA-7, and the vacuolization of LC3-GFP into punctuate bodies was determined by fluorescent microscopy. In U cells, GST-MDA-7 caused vacuolization of LC3-GFP within 24 h, effects that were also 3MA dependent (Fig. 4B; Table 3). Identical data to that in U cells were obtained in GBM6 cells with respect to GST-MDA and 3MA-dependent vacuolization of LC3-GFP (Fig. 4B; Table 3). Transfection with GFP alone did not generate punctuate bodies after GST-MDA-7 treatment (data not shown).

Considering that GST-MDAinduced cell killing appeared to cause vacuolization, which also contained putative autophagic vacuoles, we explored whether these events were dependent on the function of PERK. GST-MDA-7 induced punctuate staining of GFP-LC3 vacuoles in U and GBM6 cells within 24 h that were blocked by transient transfection of dnPERK [Fig. 4C, (i); Table 3]. Based on these findings, we determined whether knockdown of ATG5 or Beclin-1, proteins that are known to play a regulatory role in autophagy, altered GST-MDAinduced vacuole formation. The ATGATG5 and the ATG8 (LC3)-PE conjugation systems are interdependent, and a disruption in one system has a direct negative effect on the autophagic process (30&#x;33). Beclin-1 is a functional component of the lipase signaling complex, which is essential for the induction of autophagy (30&#x;33). Therefore, perturbation of the levels of ATG5 or Beclin-1 should result in reduced autophagy and the attenuation of the biological effects of GST-MDA To test this, RNA interference was used to specifically suppress ATG5 or Beclin-1 protein levels in tumor cells. Knockdown of ATG5 or Beclin-1 expression significantly suppressed GST-MDAinduced GFP-LC3 vacuolization in U cells [Fig. 4C, (ii); Table 4]. In agreement with these findings, treatment of U cells with GST-MDA-7 caused increased expression of ATG5 and Beclin-1 within 24 h as well as the cleavage of endogenous LC3 protein [Fig. 4C, (ii)]. In transformed fibroblasts, treatment with GST-MDA-7 also caused increased expression of ATG5, a more modest increase in Beclin-1 levels, and modification of endogenous LC3 protein, effects that were abolished in PERK&#x;/&#x; cells (Fig. 4D). Collectively, these data argue that GST-MDA-7 causes an initial autophagic response in human glioma cells and transformed rodent fibroblasts in vitro.

Based on the findings in Fig. 4, we determined whether modulation of PERK function or ER stress signaling altered GBM cell survival after GST-MDA-7 exposure. Overexpression of the MDA-7/IL and PERK-binding protein BiP/ GRP78 significantly suppressed LC3-GFP vacuolization after GST-MDA-7 exposure and suppressed GST-MDA-7 toxicity by 64 ± % (±SE; n = 3; Fig. 5A, top), that is, overexpressed exogenous BiP/GRP78 bound to GST-MDA-7 inside the cell, and thus lowered the free intracellular concentration of GST-MDA-7, reducing the overall level of cell killing.