Z-LEHD-FMK

Rabdocoestin B exhibits antitumor activity by inducing G2/M phase arrest and apoptosis in esophageal squamous cell carcinoma

Jingnan Wang1· Zhirong Zhang1· Yun Che1· Zuyang Yuan1· Zhiliang Lu1· Yuan Li1· Jun Wan2· Handong Sun2· Zhaoli Chen1· Jianxin Pu2· Jie He1

Abstract

Purpose Esophageal squamous cell carcinoma (ESCC) is one of the most aggressive squamous cell carcinomas and is generally resistant to chemotherapy. In the present study, the cytotoxic activity of Rabdocoestin B (Rabd-B) against ESCC and the underlying mechanisms were investigated.
Methods The inhibitory effect of Rabd-B on KYSE30 and KYSE450 was evaluated by Cell Counting Kit-8 (CCK8) and colony formation assays in vitro. The cell cycle distribution and apoptosis of cells treated with Rabd-B were determined by flow cytometry. The mechanisms underlying the effects of Rabd-B were systematically examined by Western blot. The in vivo anti-tumor ability of Rabd-B was measured in mouse xenograft models and cisplatin (DDP) was used as positive control. Results Rabd-B efficiently induced G2/M phase arrest in ESCC cells by upregulating the Chk1/Chk2-Cdc25C axis to inhibit the G2→M transition facilitated by Cdc2/Cyclin B1. Furthermore, Rabd-B suppressed ATM/ATR phosphorylation, thereby inhibiting BRCA1-mediated DNA repair, which resulted in mitotic catastrophe and induced cell apoptosis. Rabd-B also decreased the activity of the Akt and NF-κB survival signaling pathways and ultimately initiated the caspase-9-dependent intrinsic apoptotic pathway in ESCC cells. The apoptosis induced by Rabd-B could be partially reversed by a caspase9-specific inhibitor (Z-LEHD-FMK) and a pan-caspase inhibitor (Z-VAD-FMK). Moreover, Rabd-B effectively suppressed tumor growth in mouse xenografts which was comparable to that of DDP without significant injuries to the mice.
Conclusion Taken together, these findings indicate that Rabd-B is a promising precursor compound that may be useful as a treatment for ESCC and thus warrants further investigation.

Keywords Rabdocoestin B · G2/M phase arrest · Apoptosis · Cytotoxicity · Esophageal squamous cell carcinoma

Introduction

Esophageal squamous cell carcinoma (ESCC) is one of the most aggressive squamous cell carcinomas and has a high prevalence in Asia [1]. The 5-year survival rate of patients with ESCC is only 15–25% [2]. A majority of cancer patients eventually relapse or develop chemoresistance despite initial response [3]. Therefore, new drugs with greater efficacy are urgently needed for clinical treatment.
Isodon species are popular in Chinese folk medicine because these plants have various pharmacological activities, including antitumor, anti-inflammatory, and antibacterial properties [4, 5]. The compounds extracted from species of the Isodon genus have become promising sources of novel chemotherapeutic or chemopreventive agents [6–8]. Rabdocoestin B (Rabd-B, Fig. 1a) is a diterpenoid compound isolated from the leaves of Isodon serra (Maxim.) Hara (Labiatae) [9–11], and previous studies have demonstrated that Rabd-B has biological activity against cancer cells in vitro. Feng et al. demonstrated that Rabd-B is a potential proteasome inhibitor and can induce apoptosis in t(8;21) leukemia cells [12]. Another study showed that Rabd-B could inhibit cell proliferation and induce apoptosis in lung cancer cells by activating endogenous and exogenous apoptosis pathways [13]. However, the in vivo effectiveness of Rabd-B and the mechanisms underlying its effects in ESCC are unclear and require further exploration.
In the present study, we observed that Rabd-B inhibited both in vivo and in vitro proliferation of ESCC. Rabd-B initiated the Chk1/2-Cdc25C axis to induce G2/M phase arrest, and simultaneously prevented DNA repair by downregulating ATM/ATR-BRCA1 activity. Furthermore, Rabd-B inhibited the Akt and the NF-κB signaling pathways. Ultimately, Rabd-B triggered caspase-9-regulated apoptosis in ESCC cells. Moreover, Rabd-B effectively suppressed tumor growth in mouse xenografts which was comparable to that of cisplatin (DDP) without significant injuries to the mice.

Materials and methods

Chemicals and antibodies

Rabd-B was isolated from the leaves of Isodon serra as described previously [14]. The purifying analysis of Rabd-B was performed on an Agilent 1200 liquid chromatography (Agilent Technologies, Palo Alto, CA, USA) with a Zorbax SB-C18 (4.6 mm × 250 mm) column (10–100% CH3CN:H2O, 30 min, 238 nm, 1 mL/min) with purity more than 98%. The structure of Rabd-B (Online Resource: Spectroscopic data of Rabd-B and Fig. S1-3) was confirmed by comparison of the NMR and HREISMS data with published data [14].
Rabd-B powder was dissolved in dimethyl sulfoxide (DMSO) (D2650, Sigma) as a 250 mM solution and stored at − 20 °C. Cisplatin (P4394, Sigma) was dissolved in 0.9% w/v NaCl solution and stored at − 20 °C, and 10% Pluronic F68 (#24040032) was purchased from Life
Technologies Corporation. Caspase-9 inhibitor (Z-LEHDFMK, B3233), caspase-8 inhibitor (Z-IETD-FMK, B3232) and pan-caspase inhibitor (Z-VAD-FMK, A1902) was purchased from APExBIO (ApexBio Technology). DNA Damage Antibody Sampler Kit (#9947), Apoptosis Antibody Sampler Kit (#9915), Phospho-Akt Pathway Antibody Sampler Kit (#9916), NF-κB Pathway Antibody Sampler Kit (#9936), Cdc25C Antibody Sampler Kit (#9555), Caspase-8 Antibody (#9746), Bax Antibody (#2772), Bcl-2 Antibody (#2870), Cdc2 Antibody (#9112), Phospho-Cdc2 Antibody (#4539), Cyclin B1 (#4138), Ki67 (IHC Specific, #9027), GAPDH Antibody (#5174), Anti-rabbit IgG (HRP-linked Antibody, #7071) and Antimouse IgG (HRP-linked Antibody, #7072) were purchased from CST (Cell Signaling Technology). All experiments, with the exception of the in vivo antitumor assays, were repeated triplicate.

Cell lines and animals

Seven esophageal cancer cell lines (KYSE30, KYSE450, KYSE70, KYSE150, KYSE180, KYSE410 and KYSE510) and the non-transformed NIH-3T3 cells were cultured in a 37 °C incubator (Thermo, Rockford, IL, USA) supplied with 5% CO2. The culture medium was RPMI 1640 (Corning, Logan, UT, Japan) supplemented with 10% fetal bovine serum (Corning, Mediatech Inc., Australia) and 1% penicillin–streptomycin (HyClone, Logan, UT, USA). The ESCC cell lines (KYSE series) were established from samples of human ESCC patients by Dr. Yutaka Shimada [15, 16]. All the cell lines underwent STR analysis and were matched to the existing reference genotypes.
The animal experiments were approved by the Cancer Institute and Hospital of the Chinese Academy of Medical Sciences Institutional Animal Care and Use Committee. All procedures performed in studies involving animals were in accordance with the ethical standards. Athymic nude mice (females, 3–4-weeks-old, 13–17 g) were purchased from H.F.K. Bioscience Co., Ltd. (Beijing, China). The animals were fed a standard commercial diet produced by the Experimental Animal Center of the Chinese Academy of Medical Sciences and maintained under pathogen-free conditions and a 12-h light–dark cycle.

Cell viability detection

A Cell Counting Kit-8 (CCK8) (Dojindo, Kumamoto, Japan) was used to test the proliferative abilities of the cells. Briefly, 2 × 103 cells were seeded in 96-well plates and treated with different concentrations of Rabd-B for 12, 24, 48, and 72 h. CCK8 reaction signals were measured by a SpectraMax® 190 (Molecular Device, Sunnyvale, CA, USA) at 450 nm. Colony formation assays were conducted in 6-well plates, each of which contained 500 cells that were treated with Rabd-B or 0.1% DMSO for 24 h and incubated in no-treatment culture medium for another 10 days.

Cell cycle analysis

A total of 3 × 105 cells were seeded in each 6-cm dish before incubation overnight and synchronization for 12 h in serumfree medium. Then, the cells were incubated in complete culture medium containing Rabd-B for 24 h. The cells were collected, fixed with 70% alcohol at 4 °C overnight, digested in RNase at 37 °C for 30 min, stained with propidium iodide (PI) for 20 min while protected from light [8], and analyzed with a BD Flow Cytometer (Becton Dickinson FACSCanto II).

Cell apoptosis detection

A BD Pharmingen FITC Annexin V Apoptosis Detection Kit (BD Biosciences, NJ, USA) was used to detect cell apoptosis. Briefly, after Rabd-B or mock treatment, the cells were harvested, resuspended at a concentration of 1 × 106 cells/ mL, and stained with FITC Annexin V and PI in the dark. Cell apoptosis was assessed by a BD flow cytometer within 1 h of the experiment.

Western blotting (WB)

The cells were lysed in RIPA buffer in the presence of protease and phosphatase inhibitors (Thermo). After quantification with a BCA Protein Assay Kit (Thermo), 20 μg of protein was separated by SDS–PAGE and transferred to PVDF membranes (Millipore, Billerica, MA, USA). The samples were then incubated with the appropriate primary and secondary antibodies, and band intensities were detected by peroxidase reactions using an ECL detection system (Millipore) [7].

Immunofluorescence microscopy

2 × 103 cells were seeded in 96-well plates overnight and treated with Rabd-B or DMSO for 24 h. After being fixed, permeabilized and blocked, the cells were labeled with the primary (antibody rabbit anti-γH2AX antibody, #9718, Cell Signaling Technology) for 3 h at room temperature. Then, secondary Antibody Alexa Fluor® 594 conjugate (A-11037, Thermo) was incubated for 45 min at room temperature. Nuclei were stained with DAPI. The images were captured at 200 × magnification by fluorescence microscopy (Nikon, Tokyo, Japan).

In vivo antitumor experiment

The mice were randomly divided into the following 3 groups (seven mice per group): a negative control group, a positive control group and a Rabd-B-treatment group. A total of 1.2 × 106 KYSE30 cells in 200 μL were transplanted into the right flank of each mouse. Treatment was initiated on the 9th day after transplantation. The mice in different groups were intraperitoneally injected with 200 μL of 1% Pluronic F68, 2 mg/kg DDP, or 12 mg/kg Rabd-B every other day. Tumors were measured every other day with a digital caliper, and the tumor volume was calculated according to the following formula: 0.5 × L × W2 [17]. At the end of the study, animals were sacrificed humanly and tumors were excised carefully and measured tumor weights.

Tumor histology and immunohistochemistry (IHC)

Tumor xenograft tissue samples were fixed in formalin and embedded in paraffin. Tumor histology was analyzed by hematoxylin and eosin (H&E), and Ki67 expression levels were assessed by IHC, as described previously [7].

Terminal deoxynucleotide transferase‑mediated dUTP end labeling (TUNEL) assay

The apoptotic signals of the xenograft tissues were detected by a DeadEnd™ Fluorometric TUNEL System (Promega, Madison, WI, USA). Briefly, the tissue sections were deparaffinized and rehydrated before fixation in paraformaldehyde and digestion with Proteinase K solution. The slides were subsequently incubated with rTdT incubation buffer at 37 °C for 60 min before undergoing a finishing reaction in 2 × SCC. The nuclei were stained using DAPI, and the signals were visualized by fluorescence microscopy (Nikon, Tokyo, Japan) [18].

Statistical analyses

GraphPad Prism 5 (GraphPad Software Inc., San Diego, CA, USA) was used to analyze the significance of the differences among the groups. One-way ANOVA was performed for comparisons among three groups, and Student’s t test was performed to analyze the differences between two independent groups. The data are expressed as the mean ± SEM for the in vitro study results and as the mean ± SD for the in vivo study results [8]. p < 0.05 was considered statistically significant. Results Rabd‑B suppresses cell growth and colony formation in ESCC cell lines Firstly, seven ESCC cell lines and the non-transformed NIH3T3 cell line were used for proliferation inhibition screening of Rabd-B. The results showed that Rabd-B exhibited highly inhibition rate to ESCC cell lines. Meanwhile, NIH3T3 cells were much more resistant to Rabd-B treatment (Online Resource: Fig. S4). Two ESCC cell lines, KYSE30 and KYSE450, were used for further investigation. The results (Fig. 1b, c) showed that Rabd-B significantly inhibited KYSE30 and KYSE450 cell proliferation, with I C50 values of 1.56 and 1.94 μM at 72 h, respectively. Furthermore, the colony formation assay results indicated that Rabd-Btreated KYSE30 and KYSE450 cells displayed both fewer and smaller colonies than those of DMSO-treated control cells (Fig. 1d). These results indicate that Rabd-B displayed significant cytotoxicity in ESCC cell lines in vitro. In addition, the non-transformed cell line NIH-3T3 was more tolerant to Rabd-B treatment (Online Resource: Fig. S5). Rabd‑B triggers cell cycle arrest at G2/M phase through a Chk1/2‑mediated pathway and prevents DNA repair by suppressing ATM/ATR‑BRCA1 activity The post-Rabd-B treatment cell cycle distributions of KYSE30 and KYSE450 cells were examined by flow cytometry. As shown in Fig. 2a, the percentage of cells in G2/M phase was increased in the group treated with 3 μM Rabd-B for 24 h compared with that of the negative control group (KYSE30, 44.4 vs 30.9%, p < 0.001; KYSE450, 37.9 vs 32.1%, p < 0.05), and the numbers of cells that experienced arrest continued to increase with 6 μM Rabd-B treatment (KYSE30, 56.6 vs 30.9%, p < 0.001; KYSE450, 44.7 vs 32.1%, p < 0.001). Since Rabd-B-treated cells exhibited an apparent G2/M delay, the possible mechanisms responsible for causing DNA damage and regulating the G2/M phase transition were investigated. Phosphorylated histone H2AX (γH2AX) is a sensitive surrogate marker of DNA double-stranded breaks (DSB) and plays an important role in DNA damage response (DDR) [19, 20]. To demonstrate the DNA damage response, immunofluorescence staining was performed in Rabd-B treated cells and DMSO control cells to evaluate the γH2AX foci. As shown in Fig. 2b, KYSE30 and KYSE450 cells experienced significant DNA damage as indicated by the significant γH2AX foci in most of the cells treated with DDP and Rabd-B. There were no detectable γH2AX foci in DMSO-control cells. WB confirmed the DSB lesion of cells treated with Rabd-B as manifested by increased expression of γH2AX in response to escalating Rabd-B concentrations (Fig. 2c). As consequences, p-Chk2 (Thr68) expression increased significantly, and p-Chk1 (Ser345) levels first increased and then decreased. Cdc25C and p-Cdc25C (Ser216) expression levels decreased concurrent with increases in Chk1/2 activity. The results also showed that Cdc2 phosphorylation (Tyr15) and Cdc2 and Cyclin B1 expression decreased significantly with increasing Rabd-B concentrations (Fig. 2c). The above results indicated that the DNA damage initiated by Rabd-B triggered Chk1 and Chk2 activation, which inhibited Cdc25C, resulting in Cdc2/Cyclin B1 inactivation and G2/M arrest. The ATM/ATR-BRCA1 pathway plays an important role in the DNA homologous recombination repair (HR) [21]. As a target of ATM/ATR, BRCA1 can be phosphorylated at Ser1524 [21, 22]. In the present study, the expression of p-ATM (Ser1981) and p-ATR (Ser428) was significantly decreased in response to Rabd-B treatment, which indicated that ATM/ATR activation were suppressed by RabdB. BRCA1 phosphorylation at site Ser1524 was downregulated along with ATM/ATR inactivation (Fig. 2d). Thus, the ATM/ATR-BRCA1 pathway was suppressed by RabdB, which may lead to repair dysfunction and promote cell apoptosis. Rabd‑B induces ESCC cell apoptosis via the caspase‑9‑dependent intrinsic pathway The apoptosis of ESCC cells after Rabd-B treatment was evaluated by flow cytometry analyses. As shown in Fig. 3a, following a 24-h reaction with 4 or 8 μM Rabd-B, the proportions of apoptotic cells increased in a dose-dependent manner in the treated groups compared with those of the control group (KYSE30, 4 μM: 6.3 vs 1.4%, 8 μM: 14.4 vs 1.4%, both p < 0.001; KYSE450, 4 μM: 4.8 vs 1.9%, 8 μM: 7.3 vs 1.9%, both p < 0.001). A progressive increase in the apoptotic cell population occurred in the groups of cells exposed to Rabd-B for 48 h compared with that of the groups of cells exposed to the control treatment for the same period of time (KYSE30, 4 μM: 15.5 vs 3.8%, 8 μM: 27.2 vs 3.8%, both p < 0.001; KYSE450, 4 μM: 21.2 vs 1.8%, 8 μM: 36.4 vs 1.8%, both p < 0.001) (Fig. 3a). In contrast, Rabd-B had little effect on the apoptosis of the normal NIH-3T3 cells (Online Resource: Fig. S6). The expression of a series of proteins correlated with apoptosis was then analyzed by WB to evaluate the intracellular alterations of ESCC cells exposed to Rabd-B. As shown in Fig. 3b, along with increased Rabd-B concentrations, cleaved caspase-9, cleaved caspase-3 and cleaved PARP expression levels increased gradually. However, cleaved caspase-8 exhibited only limited expression, which was unaltered by Rabd-B treatment, and the mitochondrial pathway regulators Bax and Bcl-2 displayed no changes in expression. These results indicated that Rabd-B induced apoptosis via a caspase-9-dependent intrinsic apoptotic pathway independent of Bax and Bcl-2. The apoptosis induced by Rabd-B could be partially reversed by a caspase-9-specific inhibitor (Z-LEHD-FMK) and a pan-caspase inhibitor (Z-VAD-FMK) but was not reversed by a caspase-8 inhibitor (Z-IETD-FMK) (Fig. 4a, b). In addition, the caspase-9 and pan-caspase inhibitors at least partially protected procaspase-9, procaspase-3 and PARP from cleavage (Fig. 4c). These results confirmed that Rabd-B-induced apoptosis mainly via a caspase-9-dependent intrinsic apoptotic pathway. Rabd‑B significantly inhibits the Akt and NF‑κB signaling pathways, resulting in cell apoptosis The above results showed that Rabd-B induces ESCC cell apoptosis through a caspase-9-dependent pathway, but the process by which this pathway is regulated remains unclear. The Akt and NF-κB signaling pathways are crucial for ensuring cell survival [23, 24]. Therefore, the effects of Rabd-B on these survival signaling pathways were systematically examined. As shown in Fig. 5, a significant decrease in p-Akt (Ser473) levels and a slight downregulation of total Akt levels were observed, which indicated that Rabd-B suppressed the Akt signaling pathway predominantly by inhibiting Akt phosphorylation at residue Ser473. The expression levels of IKKα and IKKβ, which function as activators of NF-κB signaling, gradually decreased, while those of IκBα, an NF-κB inhibitor, increased concurrently with increasing concentrations of Rabd-B. The expression levels of NF-κB in KYSE30 gradually dropped along with climbing Rabd-B concentration, while in KYSE450, NF-κB seemed unchanged (Fig. 5). There were only trace levels of p-IKKα/β, p-IκBα and p-NF-κB expression and the change tendency of these proteins remained ambiguous (Fig. 5). These findings demonstrated that Rabd-B mainly influenced Akt signaling pathway by inhibiting the phosphorylation of p-Akt (Ser473) and NF-κB signaling pathway by suppressing the expression of IKKα and IKKβ and enhancing IκBα. As consequences, both the Akt and NF-κB signaling pathways were significantly inhibited by Rabd-B. Rabd‑B induces tumor regression in mouse xenograft models Mouse xenograft models were used to evaluate the in vivo antitumor efficacy of Rabd-B. Rabd-B efficiently suppressed tumor growth in the treated groups compared with that of the negative control group (Fig. 6a–c). The mean tumor volume increased from 28.02 ± 9.63 to 1368.61 ± 323.07 mm3 in the negative control group, whereas the mean tumor volume increased from 31.92 ± 17.96 to 564.44 ± 115.84 mm3 and from 34.65 ± 23.60 to 595.54 ± 141.33 mm3 in the Rabd-Btreated group and DDP-treated group, respectively (Fig. 6b). Tumor weights were significantly lighter in the Rabd-B- and DDP-treated groups than those in the control group, with weights of 0.42 ± 0.17, 0.38 ± 0.12 and 1.01 ± 0.37 g, respectively (Fig. 6c). Mouse body weight surveillance revealed no difference in body weight among the three treatment groups. As shown in Fig. 6d, no significant body weight loss was observed in any of the groups. Furthermore, no significant injuries to the liver or kidney occurred in Rabd-B- and DDP-treated mice (Fig. 6e). In tumor tissues, apparent histopathological disorders, such as large vacuolization, cell debris and necrosis nests, were observed in Rabd-B- and DDP-treated groups, whereas viable tumor cell nests without significant cell death were observed in the negative-control samples as demonstrated by H&E staining (Fig. 6f). Proliferation was assessed by Ki67 expression, and apoptotic signals were measured by TUNEL staining in the tumor tissues of different treatment groups. As shown in Fig. 6f, the negative control samples exhibited strong nuclear Ki67 expression, whereas the Rabd-B- and DDP-treated groups exhibited barely detectable Ki67 expression. Accordingly, significant apoptosis signals were detected by TUNEL assays in the Rabd-B- and DDP-treated groups, whereas rare apoptosis signals were detected in the negative control group (Fig. 6g). Discussion ESCC is an intractable malignant with a low 5-year survival rate of patients [2]. Nowadays, DDP and 5-Fu are the most widely used chemotherapeutic agents for ESCC. However, the median survival time remains unsatisfactory [25]. ESCC patients usually have a poor prognosis partially due to less sensitive to chemotherapy agents, so exploration of new drugs or treatment strategies for combating resistance has important significance. Diterpenoids are important chemicals isolated from natural products and some of the compounds have been reported the anti-ESCC potential. Oridonin, a diterpenoid isolated from Isodon rubescens, was reported as an active diterpenoid which effectively induced cell cycle arrest, apoptotic and autophagic pathways for cancer therapeutics [26]. Recently, Pi et al. discovered that oridonin was a strong anticancer agent targeting EGF-EGFR interaction in ROS-dependent mechanism in ESCC cells [27]. As an effective agent, oridonin was injected alone or in combination with other chemotherapy drugs for the treatment of liver cancer and gastric cardia carcinoma in China [28]. Studies later discovered a novel diterpenoid named jaridonin, was more potent than oridonin in inhibiting proliferation and pro-apoptotic in esophageal cancer cell lines [29, 30]. Wang et al. showed another diterpenoid jesridonin has extensive anti-tumor activity. Moreover, jesridonin in combination with paclitaxel has synergistic cytotoxic effects on human esophageal carcinoma [31, 32]. In view of the limited effectiveness of current ESCC therapies and the potential of diterpenoids isolated from natural products [6, 8], such compounds may serve as a fertile source of novel candidate molecules for anti-ESCC drug discovery. In this study, we found that Rabd-B isolated from Isodon serra effectively inhibited the proliferation and colony growth of ESCC cells in vitro and exhibited antitumor activity comparable to that of DDP in vivo with relative safety. The underlying mechanisms of the antitumor activity were systematically investigated. Genotoxic chemicals can attack DNA and produce a variety of DNA lesions [19]. γH2AX, a marker for DNA damage, is an early indicator of DSB and plays an important role in DDR [20]. DDRs are orchestrated by multiple signal transduction processes, including the Chk1/Chk2 checkpoint pathway [33, 34]. Chk1 and Chk2 are checkpoint kinases involved in the G2/M phase regulation [33, 34]. When activated in response to DNA damage, Chk1/2 negatively regulates Cdc25C, resulting in inactivation of Cdc2 and G2/M arrest [34, 35]. In this study, increasing γH2AX expression indicated that significant DNA damage occurred following Rabd-B treatment. The increases in p-Chk1 (Ser345) and p-Chk2 (Thr68) indicated the initiation of G2/M phase regulation, resulting in Cdc25C inhibition. We also observed evident decreases in Cdc2, p-Cdc2 (Tyr15) and Cyclin B1, consistent with the pattern of Cdc25C expression. As the downstream target of Cdc25C [36, 37], Cdc2 was inhibited by Cdc25C inactivation, resulting in G2/M arrest. Cyclin B1 expression was also downregulated by Rabd-B to enhance the G2/M phase transition delay. Cells initiate cell cycle arrest to allow for DNA injury repair. BRCA1—a pleiotropic DNA damage response protein—is generally activated by ATM/ATR and functions in HR for high-fidelity repair of DNA DSBs [21]. In the present study, ATM, ATR and BRCA1 were suppressed simultaneously in response to Rabd-B treatment, indicating that BRCA1 inactivation was ATM/ATR-dependent. BRCA1 inactivation led to repair dysfunction and promoted premature entry into mitosis, resulting in mitotic catastrophe [33]. Thus, cells may switch off repair and stimulate cell death to eliminate cells with DNA damage [19]. The Akt and NF-κB signaling pathways are crucial for ensuring cell survival [23, 24]. The Akt pathway modulates many cellular processes and is frequently altered in cancer, contributing to tumor growth and survival [38]. Small molecule inhibitors targeting the Akt signaling pathway have shown promising preclinical activity [23]. The NF-κB pathway, a critical regulator of apoptosis, plays an important role in the development and progression of cancer and chemoresistance [39]. Activation of NF-κB is frequently observed in tumor cells, and this pathway has emerged as a promising anti-cancer target [40, 41]. Blocking the Akt and NF-κB pathways may trigger cell apoptosis and could be used for prevention and treatment of malignancies [23, 40, 42]. Previous studies have reported aberrantly high Akt [43–45] and NF-κB [42, 46, 47] signaling in ESCC. The highly activated Akt [43–45] and NF-κB [42, 46, 47] signals in ESCC mediate diverse pro-survival signals, promote the malignant phenotype of cancer cells, and correlate with poor prognosis. In this study, we found that the Akt and NF-κB pathways were constitutively activated in untreated KYSE30 and KYSE450 cells, indicating their role in promoting ESCC cell survival. Rabd-B treatment suppressed the Akt signaling pathway mainly by inhibiting Akt phosphorylation. Simultaneously, Rabd-B significantly decreased NF-κB pathway activity by inhibiting the expression of the signaling activators IKKα and IKKβ and inducing the expression of the signaling inhibitor IκBα. Taken together, two pivotal survival pathways, namely, the Akt and NF-κB signaling pathways were significantly inhibited by Rabd-B, driving cells into the apoptotic progress. Apoptosis is mediated by intracellular cysteine proteases called caspases, which can be classified as upstream initiators or downstream effectors of apoptosis [48, 49]. In the intrinsic pathway, the apoptosis initiator caspase-9 is activated, and caspase-8 is activated in the extrinsic pathway [48, 50]. Activation of either caspase-9 or caspase-8 can mediate activation of the effector caspase-3, resulting in apoptotic cascade amplification and cell death [19, 48]. The present study showed that Rabd-B induces apoptosis of ESCC cells through a caspase-9-dependent intrinsic apoptotic pathway independent of Bax and Bcl-2. Moreover, the apoptotic process could be protected by caspase-9 and pancaspase inhibitors. Thus far, there is no evidence for the antitumor effects of Rabd-B in vivo. In this study, administration of Rabd-B produced a similar inhibitory effect on KYSE30 cell proliferation as that of DDP. Ki67 expression is strongly associated with tumor cell proliferation [51], and the TUNEL staining indicates cell apoptosis [52, 53]. In tumor xenograft tissues, significant TUNEL staining, but almost no Ki67 expression was detected in the Rabd-B- and DDP-treated groups, indicating significant tumor growth suppression and cytotoxic effects of Rabd-B in vivo. Furthermore, there were virtually no effects on the mouse weight and no toxicity to the liver and kidney. These findings suggest that Rabd-B is a relatively efficacious and safe agent targeting ESCC. Taken together, the results showed that Rabd-B Z-LEHD-FMK is a promising compound that may be a useful and relatively safe chemotherapy agent for ESCC and thus warrants further research and development as an anti-cancer treatment.

References

1. Ohashi S, Miyamoto S, Kikuchi O, Goto T, Amanuma Y, Muto M (2015) Recent advances from basic and clinical studies of esophageal squamous cell carcinoma. Gastroenterology 149(7):1700–1715. https://doi.org/10.1053/j.gastro.2015.08.054
2. Pennathur A, Gibson MK, Jobe BA, Luketich JD (2013) Oesophageal carcinoma. Lancet 381(9864):400–412. https:// doi.org/10.1016/S0140-6736(12)60643-6
3. Rustgi AK, El-Serag HB (2014) Esophageal carcinoma. N Engl J Med 371(26):2499–2509. https://doi.org/10.1056/NEJMra1314530
4. Sun HD, Huang SX, Han QB (2006) Diterpenoids from Isodon species and their biological activities. Nat Prod Rep 23(5):673–698. https://doi.org/10.1039/b604174d
5. Peters RJ (2010) Two rings in them all: The labdane-related diterpenoids. Nat Prod Rep 27(11):1521–1530. https://doi. org/10.1039/c0np00019a
6. Ding Z, Lao Y, Zhang H, Fu W, Zhu L, Tan H, Xu H (2016) Griffipavixanthone, a dimeric xanthone extracted from edible plants, inhibits tumor metastasis and proliferation via downregulation of the RAF pathway in esophageal cancer. Oncotarget 7(2):1826–1837. https://doi.org/10.18632/oncotarget.6484
7. Liao YJ, Bai HY, Li ZH, Zou J, Chen JW, Zheng F, Zhang JX, Mai SJ, Zeng MS, Sun HD, Pu JX, Xie D (2014) Longikaurin A, a natural ent-kaurane, induces G2/M phase arrest via downregulation of Skp2 and apoptosis induction through ROS/JNK/cJun pathway in hepatocellular carcinoma cells. Cell Death Dis 5:e1137. https://doi.org/10.1038/cddis.2014.66
8. Yao R, Chen Z, Zhou C, Luo M, Shi X, Li J, Gao Y, Zhou F, Pu J, Sun H, He J (2015) Xerophilusin B induces cell cycle arrest and apoptosis in esophageal squamous cell carcinoma cells and does not cause toxicity in nude mice. J Nat Prod 78(1):10–16. https://doi.org/10.1021/np500429w
9. Wan J, Liu M, Jiang HY, Yang J, Du X, Li XN, Wang WG, Li Y, Pu JX, Sun HD (2016) Bioactive ent-kaurane diterpenoids from Isodon serra. Phytochemistry 130:244–251. https://doi. org/10.1016/j.phytochem.2016.05.014
10. Xu Y, Kubo I (1993) Diterpenoid constituents from Rabdosia coetsa. Phytochemistry 34(2):576–578. https://doi. org/10.1016/0031-9422(93)80051-S
11. Wan J, Jiang HY, Tang JW, Li XR, Du X, Li Y, Sun HD, Pu JX (2017) Ent-Abietanoids Isolated from Isodon serra. Molecules 22 (2). https://doi.org/10.3390/molecules22020309
12. Feng T, Pu J, Hu Z, Liu D, Sun H, Zhou G (2009) Rabdocoetsin B, a diterpenoid isolated from Isodon coetsa, is a potential proteasome inhibitor and induced apoptosis of t(8;21) leukemia cells. Sheng Wu Gong Cheng Xue Bao 25(8):1218–1224
13. Ma L, Wen ZS, Liu Z, Hu Z, Ma J, Chen XQ, Liu YQ, Pu JX, Xiao WL, Sun HD, Zhou GB (2011) Overexpression and small molecule-triggered downregulation of CIP2A in lung cancer. PLoS One 6(5):e20159. https://doi.org/10.1371/journal. pone.0020159
14. Wang XR, Wang ZQ, Wang HP, Hu HP, Wang DQ (1987) Chemical structures of Coetsin A and B. Zhiwu Xuebao 29(4):412–415
15. Shimada Y, Imamura M, Wagata T, Yamaguchi N, Tobe T (1992) Characterization of 21 newly established esophageal cancer cell lines. Cancer 69(2):277–284
16. Kanda Y, Nishiyama Y, Shimada Y, Imamura M, Nomura H, Hiai H, Fukumoto M (1994) Analysis of gene amplification and overexpression in human esophageal-carcinoma cell lines. Int J Cancer 58(2):291–297
17. Shen K, Xie J, Wang H, Zhang H, Yu M, Lu F, Tan H, Xu H (2015) Cambogin induces caspase-independent apoptosis through the ROS/JNK pathway and epigenetic regulation in breast cancer cells. Mol Cancer Ther 14(7):1738–1749. https:// doi.org/10.1158/1535-7163.MCT-14-1048
18. Kumar D, Das B, Sen R, Kundu P, Manna A, Sarkar A, Chowdhury C, Chatterjee M, Das P (2015) Andrographolide analogue induces apoptosis and autophagy mediated cell death in U937 cells by inhibition of PI3K/Akt/mTOR pathway. PLoS One 10(10):e0139657. https://doi.org/10.1371/journal.pone.0139657
19. Roos WP, Thomas AD, Kaina B (2016) DNA damage and the balance between survival and death in cancer biology. Nat Rev Cancer 16(1):20–33. https://doi.org/10.1038/nrc.2015.2
20. Benzina S, Pitaval A, Lemercier C, Lustremant C, Frouin V, Wu N, Papine A, Soussaline F, Romeo PH, Gidrol X (2015) A kinome-targeted RNAi-based screen links FGF signaling to H2AX phosphorylation in response to radiation. Cell Mol Life Sci 72(18):3559–3573. https://doi.org/10.1007/s00018-015-1901-7
21. Roy R, Chun J, Powell SN (2011) BRCA1 and BRCA2: different roles in a common pathway of genome protection. Nat Rev Cancer 12(1):68–78. https://doi.org/10.1038/nrc3181
22. Shechter D, Costanzo V, Gautier J (2004) Regulation of DNA replication by ATR: signaling in response to DNA intermediates. DNA Repair 3(8–9):901–908. https://doi.org/10.1016/j. dnarep.2004.03.020
23. LoRusso PM (2016) Inhibition of the PI3K/AKT/mTOR pathway in solid tumors. J Clin Oncol 34:3803–3815. https://doi. org/10.1200/jco.2014.59.0018
24. Karin M (2006) Nuclear factor-kappaB in cancer development and progression. Nature 441(7092):431–436. https://doi.org/10.1038/ nature04870
25. Baba Y, Saeki H, Nakashima Y, Oki E, Shigaki H, Yoshida N, Watanabe M, Maehara Y, Baba H (2016) Review of chemotherapeutic approaches for operable and inoperable esophageal squamous cell carcinoma. Dis Esophagus. https://doi.org/10.1111/ dote.12521
26. Li CY, Wang EQ, Cheng Y, Bao JK (2011) Oridonin: an active diterpenoid targeting cell cycle arrest, apoptotic and autophagic pathways for cancer therapeutics. Int J Biochem Cell Biol 43(5):701–704. https://doi.org/10.1016/j.biocel.2011.01.020
27. Pi J, Jin H, Jiang J, Yang F, Cai H, Yang P, Cai J, Chen ZW (2017) Single molecule force spectroscopy for in-situ probing oridonin inhibited ROS-mediated EGF-EGFR interactions in living KYSE150 cells. Pharmacol Res 119:479–489. https://doi.org/10.1016/j. phrs.2016.11.036
28. Ding C, Zhang Y, Chen H, Yang Z, Wild C, Ye N, Ester CD, Xiong A, White MA, Shen Q, Zhou J (2013) Oridonin ring A-based diverse constructions of enone functionality: identification of novel dienone analogues effective for highly aggressive breast cancer by inducing apoptosis. J Med Chem 56(21):8814–8825. https://doi.org/10.1021/jm401248x
29. Ma YC, Ke Y, Zi X, Zhao W, Shi XJ, Liu HM (2013) Jaridonin, a novel ent-kaurene diterpenoid from Isodon rubescens, inducing apoptosis via production of reactive oxygen species in esophageal cancer cells. Curr Cancer Drug Targets 13(6):611–624
30. Ma YC, Su N, Shi XJ, Zhao W, Ke Y, Zi X, Zhao NM, Qin YH, Zhao HW, Liu HM (2015) Jaridonin-induced G2/M phase arrest in human esophageal cancer cells is caused by reactive oxygen species-dependent Cdc2-tyr15 phosphorylation via ATM-Chk1/2Cdc25C pathway. Toxicol Appl Pharmacol 282(2):227–236. https://doi.org/10.1016/j.taap.2014.11.003
31. Wang C, Guo L, Wang S, Wang J, Li Y, Dou Y, Wang R, Shi H, Ke Y, Liu H (2017) Anti-proliferative effect of Jesridonin on paclitaxel-resistant EC109 human esophageal carcinoma cells. Int J Mol Med 39(3):645–653. https://doi.org/10.3892/ ijmm.2017.2867
32. Wang C, Yang D, Jiang L, Wang S, Wang J, Zhou K, Shi X, Chang L, Liu Y, Ke Y, Liu H (2017) Jesridonin in combination with paclitaxel demonstrates synergistic anti-tumor activity in human esophageal carcinoma cells. Bioorg Med Chem Lett 27(9):2058–2062. https://doi.org/10.1016/j.bmcl.2017.02.008
33. Smith J, Tho LM, Xu N, Gillespie DA (2010) The ATM-Chk2 and ATR-Chk1 pathways in DNA damage signaling and cancer. Adv Cancer Res 108:73–112. https://doi.org/10.1016/ B978-0-12-380888-2.00003-0
34. Reinhardt HC, Yaffe MB (2009) Kinases that control the cell cycle in response to DNA damage: Chk1, Chk2, and MK2. Curr Opin Cell Biol 21(2):245–255. https://doi.org/10.1016/j. ceb.2009.01.018
35. Sun WJ, Huang H, He B, Hu DH, Li PH, Yu YJ, Zhou XH, Lv Z, Zhou L, Hu TY, Yao ZC, Lu MD, Shen X, Zheng ZQ (2017) Romidepsin induces G2/M phase arrest via Erk/cdc25C/cdc2/ cyclinB pathway and apoptosis induction through JNK/c-Jun/ caspase3 pathway in hepatocellular carcinoma cells. Biochem Pharmacol 127:90–100. https://doi.org/10.1016/j.bcp.2016.12.008
36. Gavet O, Pines J (2010) Progressive activation of CyclinB1-Cdk1 coordinates entry to mitosis. Dev Cell 18(4):533–543. https://doi. org/10.1016/j.devcel.2010.02.013
37. Bulavin DV, Amundson SA, Fornace AJ (2002) p38 and Chk1 kinases: different conductors for the G(2)/M checkpoint symphony. Curr Opin Genet Dev 12(1):92–97
38. Dienstmann R, Rodon J, Serra V, Tabernero J (2014) Picking the point of inhibition: a comparative review of PI3K/AKT/mTOR pathway inhibitors. Mol Cancer Ther 13(5):1021–1031. https:// doi.org/10.1158/1535-7163.MCT-13-0639
39. Yu L, Li L, Medeiros LJ, Young KH (2017) NF-kappaB signaling pathway and its potential as a target for therapy in lymphoid neoplasms. Blood Rev 31(2):77–92. https://doi.org/10.1016/j. blre.2016.10.001
40. Godwin P, Baird AM, Heavey S, Barr MP, O’Byrne KJ, Gately K (2013) Targeting nuclear factor-kappa B to overcome resistance to chemotherapy. Front Oncol 3:120. https://doi.org/10.3389/ fonc.2013.00120
41. Panday A, Inda ME, Bagam P, Sahoo MK, Osorio D, Batra S (2016) Transcription factor NF-kappaB: an update on intervention strategies. Arch Immunol Ther Exp (Warsz) 64(6):463–483. https://doi.org/10.1007/s00005-016-0405-y
42. Tian F, Zhang C, Tian W, Jiang Y, Zhang X (2012) Comparison of the effect of p65 siRNA and curcumin in promoting apoptosis in esophageal squamous cell carcinoma cells and in nude mice. Oncol Rep 28(1):232–240. https://doi.org/10.3892/or.2012.1777
43. Li B, Xu WW, Lam AKY, Wang Y, Hu HF, Guan XY, Qin YR, Saremi N, Tsao SW, He QY, Cheung ALM (2017) Significance of PI3K/AKT signaling pathway in metastasis of esophageal squamous cell carcinoma and its potential as a target for anti-metastasis therapy. Oncotarget. https://doi.org/10.18632/oncotarget.16333
44. Yoshioka A, Miyata H, Doki Y, Yasuda T, Yamasaki M, Motoori M, Okada K, Matsuyama J, Makari Y, Sohma I, Takiguchi S, Fujiwara Y, Monden M (2008) The activation of Akt during preoperative chemotherapy for esophageal cancer correlates with poor prognosis. Oncol Rep 19(5):1099–1107
45. Chen J, Lan T, Zhang W, Dong L, Kang N, Fu M, Liu B, Liu K, Zhang C, Hou J, Zhan Q (2015) Dasatinib enhances cisplatin sensitivity in human esophageal squamous cell carcinoma (ESCC) cells via suppression of PI3K/AKT and Stat3 pathways. Arch Biochem Biophys 575:38–45. https://doi.org/10.1016/j. abb.2014.11.008
46. Kang MR, Kim MS, Kim SS, Ahn CH, Yoo NJ, Lee SH (2009) NF-kappaB signalling proteins p50/p105, p52/p100, RelA, and IKKepsilon are over-expressed in oesophageal squamous cell carcinomas. Pathology 41(7):622–625. https://doi. org/10.3109/00313020903257756
47. Zhang L, Sun J, Zhang JQ, Yang M, Bai G, Ma XL (2014) Expression and significance of molecular biomarkers in esophageal carcinoma in different nationalities patients in Xinjiang. Genet Mol Res 13(3):5413–5425. https://doi.org/10.4238/2014.July.24.21
48. Call JA, Eckhardt SG, Camidge DR (2008) Targeted manipulation of apoptosis in cancer treatment. Lancet Oncol 9(10):1002–1011. https://doi.org/10.1016/S1470-2045(08)70209-2
49. Derakhshan A, Chen Z, Van Waes C (2017) Therapeutic small molecules target inhibitor of apoptosis proteins in cancers with deregulation of extrinsic and intrinsic cell death pathways. Clin Cancer Res 23(6):1379–1387. https://doi.org/10.1158/1078-0432.CCR-16-2172
50. Kurokawa M, Kornbluth S (2009) Caspases and kinases in a death grip. Cell 138(5):838–854. https://doi.org/10.1016/j. cell.2009.08.021
51. Li LT, Jiang G, Chen Q, Zheng JN (2015) Ki67 is a promising molecular target in the diagnosis of cancer (review). Mol Med Rep 11(3):1566–1572. https://doi.org/10.3892/mmr.2014.2914
52. Sarkissian T, Timmons A, Arya R, Abdelwahid E, White K (2014) Detecting apoptosis in Drosophila tissues and cells. Methods 68(1):89–96. https://doi.org/10.1016/j.ymeth.2014.02.033
53. Gavrieli Y, Sherman Y, Ben-Sasson SA (1992) Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J Cell Biol 119(3):493–501