Int J Clin Exp Med 2019;12(4):3476-3483www.ijcem.com /ISSN:1940-5901/IJCEM0074229

Original ArticleICG001 inhibits colon cancer growth by suppressing eIF3D signaling

Yanbo Zhang, Feng Shao, Chenggang Yang, Daogui Yang, Wenfeng Du

Department of Gastrointestinal Surgery, Liaocheng People’s Hospital, Liaocheng 252000, Shandong, China

Received February 8, 2018; Accepted October 12, 2018; Epub April 15, 2019; Published April 30, 2019

Abstract: Recently, several clinical trials have examined the efficacy of small molecule inhibitors of eukaryotic trans-lation initiation factor 3 subunit D (eIF3D) for the treatment of colorectal cancer (CRC). Unfortunately, none of these compounds has proven more effective than existing chemotherapeutic drugs, resulting in a significant unmet need for effective CRC therapies. Here, we examined the anticancer effects of ICG001, a novel eIF3D inhibitor, on CRC cells using both in vitro cell growth and apoptosis assays, as well as an in vivo HCT-116 xenograft mouse model. ICG001 induced the caspase-dependent apoptosis of CRC cells by downregulating the expression of Bcl-2 in com-bination with increased expression of Bad and Bax. Western blot analysis demonstrated that ICG001 inhibited the expression of eIF3D and its downstream targets, PI3K and AKT, in vitro. Finally, ICG001 inhibited HCT-116 xenograft growth significantly in nude mice in a dose-dependent manner. Taken together, these data suggest that ICG001 in-hibited the growth of CRC cells in vitro and in vivo via the inhibition of eIF3D signaling. These results provide a basis for further clinical investigation of ICG001 as a targeted therapy for CRC. Our findings may open a new avenue for the development of novel eIF3D inhibitors in the treatment of CRC and other cancers.

Keywords: ICG001, colon cancer, eIF3D, PI3K/AKT signaling pathway

Introduction

The incidence rate of colorectal cancer (CRC), one of the most common forms of cancer world-wide, is increasing annually [1]. Despite signifi-cant advances in the treatment of CRC, the overall survival of advanced and metastatic disease has remained virtually unchanged over the past 20 years, with the 5-year survival rate ranging from as low as 15% to nearly 90% for late- and early-stage disease, respectively [2]. There is currently no effective therapy to con-trol the recurrence and metastasis of CRC. Therefore, a better understanding of the molec-ular mechanisms underlying CRC pathology is necessary to identify new therapeutic targets that can improve the management and treat-ment of colon cancer.

A wide array of tumor suppressors and onco-genes have been identified in CRC, though sig-nificant gaps remain regarding the role of spe-cific gene alterations and their downstream functions in the initiation and progression of

CRC [3, 4]. Eukaryotic translation initiation fac-tor 3 (eIF3) is a multiprotein complex consisting of 10-13 subunits, with multiple functions in translation. Mismatches between eIF3 subuni- ts is associated with cancer progression [5, 6]. Eukaryotic translation initiation factor 3 subunit D (eIF3D), a member of the eIF3 family, plays a key role in translation initiation [7]. Recently, several studies have identified links between eIF3D and the development and progression of various tumors. Aberrant activation of eIF3D signaling due to eIF3D overexpression and/or gene mutations has been shown to play a role in the development and progression of several cancers, including breast cancer [8], prostate cancer [9], non-small cell lung cancer [10], and melanoma [11]. Zhang et al. [12] demonstrated that the overexpression of eIF3D significantly enhanced the development of gallbladder can-cer development as well as the occurrence and development of ovarian cancer. Given these strong associations, eIF3D has received signi- ficant attention as a potential therapeutic tar-get for cancer intervention.

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Figure 1. ICG001 inhibits cell proliferation and induces cell death in CRC cells. A. HCT-116 and SW480 cells were treated with ICG001 (0-2 mM) for 6 days, followed by cell counting. B. HCT-116 and SW480 cells were treated with ICG001 (0-2 mM) for 24-72 h, followed by cell viability assay (MTT as-say). All data represent the means ± SEM. (n = 3). *P < 0.05, **P < 0.01, and ***P < 0.001.

Here, we examined the anticancer effects of ICG001, a novel eIF3D inhibitor, on CRC cells using in vitro cell growth and apoptosis as- says, and an in vivo HCT-116 xenograft mou- se model. ICG001 inhibited proliferation and induced apoptosis in CRC cells in vitro. Fur- thermore, ICG001 inhibited the growth of HCT-116 xenografts in vivo, with no obvious signs of toxicity. Mechanistically, ICG001 was found to inhibit the eIF3D-mediated PI3K/AKT signal-ing pathway in CRC cells both in vitro and in vivo. Our findings suggest that ICG001 is a novel eIF3D inhibitor, which may be explored for the treatment of CRC and other tumors.

Material and methods

Cell culture

Human CRC cell lines (HCT-116 and SW480) were obtained from American Type Culture Co- llection (ATCC; Manassas, VA, USA), and cul-tured in RPMI 1640 supplemented with 10% FBS. All cell lines were cultured in a humidified atmosphere at 37°C in the 5% CO2.

Cell proliferation assay

Cell proliferation assays were conducted as described previously [13]. Briefly, cells, grown

In this party, we determined the cell apoptosis by flow cytometry. Briefly, cells were treated with various concentration of ICG001 (0-2 mM) for 72 h. And then cells were collected following staining by Annexin V-FITC Apoptosis Detection Kit I (BD Biosciences, Sparks, MD, USA). The flow cytometry was used with a FACS Calibur flow cytometer (Becton Dickinson). Cells treat-ed with DMSO alone were used as a control.

Western blot analysis

After treatment, cells were harvested and lys- ed, and protein concentrations were measured using the Bio-Rad protein assay kit (Bio-Rad, Richmond, CA, USA). Equivalent amounts of proteins (whole cell lysates) were separated on 8-15% sodium dodecyl sulfate-polyacrylamide gels and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA, USA). Membranes were blocked with 5% non-fat dry milk (dissolved in PBS containing 0.05% Tween 20) for 1 h at room temperature, and then incubated with primary antibodies over-night at 4°C, followed by probing with appropri-ate secondary antibodies conjugated to horse-radish peroxidase overnight at 4°C. Immuno- reactive bands were visualised by using Rena- issance chemiluminescence reagent (Perkin-Elmer Life Science, Boston, MA, USA).

in six-well plates, were treated with ICG001 (0-2 mM) for 6 days, followed by cell counting with a Z1 counter (Beckman Coulter, Fullerton, CA, USA). Cells treated with the vehicle (DMSO) alone served as a control.

MTT assays

For quantify the cell survival, MTT assay were performed. Briefly, cells were seeded on 96-well plates, and then tre- ated with various concentra-tion of ICG001 (0-2 mM) for 24-72 h followed by the addi-tion of 50 μl (5 mg/ml) MTT to each well and incubation for 3 h. The reaction was stopped by adding 150 μl dimethyl sulfoxide (DMSO). Finally, the absorbance was measured at 570 nm using a plate reader.

Apoptosis assay

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Figure 2. ICG001 induces apoptosis in HCT-116 cells. HCT-116 cells were treated with ICG001 (0-2 mM) for 72 h, followed by Annexin V/PI staining and flow cytometry. Representative diagrams are shown. (B) Data from (A) were statistically analysed. Means ± SEM. (n = 3). *P < 0.05, **P < 0.01.

HCT-116 xenograft model

All animal experiments carried out were appro- ved by the Animal Care and Use Committee of Liaocheng People’s Hospital. Female 4-week-old Balb/c nude mice were obtained from Bei- jing Vital River Laboratory Animal Technology Co., Ltd. HCT-116 cells (2 × 106) were injected subcutaneously into the hind flank of each mouse. When the tumours reached a volume of approximately 100 mm3, mice were randomised into four groups. ICG001 was dissolved in 5% DMSO in saline. Mice were orally treated with vehicle control, 12.5 mg/kg ICG001, 25 mg/kg ICG001, 50 mg/kg and ICG001 once daily. Tumour volume [(length × width2)/2] was deter-mined with a digital caliper. Tumour growth and mice body weight were monitored every three days. At the end of experiments, animals were killed, and the tumours were collected, photo-graphed, and analysed. And parts of tumours were evaluated by haematoxylin and eosin, and immunohistochemistry with indicated an- tibodies.

Statistical analysis

All results were expressed as mean ± SEM from at least three independent experiments. The statistical significance of mean difference be- tween two groups was determined using two-tail Student’s t-test and P values of less than 0.05 were considered significant.

Results

ICG001 inhibits cell proliferation and induced cell death in CRC cells

First of all, to assess the anticancer activity of ICG001 in CRC cells in vitro, we tested whether ICG001 inhibits cell proliferation and induces cell death in HCT-116 and SW480 cells. As shown in Figure 1A, treatment with ICG001 for 6 days inhibited cell proliferation of HCT-116 and SW480 cells in a concentration-dependent manner. Furthermore, treatment with ICG001 (0-2 mM) for 24-72 h reduced the cell viability in both HCT-116 and SW480 cells in a concen-

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tration- and time-dependent manner, as detect-ed by MTT assay (Figure 1B). Collectively, the results indicate that ICG001 is a potent anti-cancer agent for CRC cells.

ICG001 induces apoptotic cell death in CRC cells

To determine whether ICG001 induces apop-totic cell death, we performed Annexin V-FITC/PI staining, a method that is frequently used to detect apoptosis. As shown in Figure 2, treat-ment with ICG001 for 72 h induced apoptosis in HCT-116 cells in a concentration-dependent manner. ICG001 at 0.1-2 mM increased the apoptotic cells by approximately 1.3-3.3-fold, compared to the control.

To understand how ICG001 induces apoptosis, we next examined whether ICG001 alters the expression of pro-apoptotic and anti-apoptotic proteins in the cells. As shown in Figure 3,

treatment with ICG001 for 24 h markedly down-regulated the expression levels of anti-apoptot-ic proteins (Bcl-2) and meanwhile up-regulated the proapoptotic protein Bad and Bax levels in a concentration-dependent manner. Further more, we observed that ICG001 induced cas-pase-dependent apoptosis. This was consis-tent with the data that ICG001 increased the cleavage of caspase-3 and caspase-9. There- fore, our results suggest that ICG001 induces caspase-dependent apoptotic cell death by down-regulating the expression of anti-apopto- tic protein Bcl-2 and up-regulating the expres-sion of pro-apoptotic protein Bad and Bax in CRC cells.

ICG001 inhibits PI3K/AKT signaling in CRC cells

To further illuminate the molecular mechanis- ms by which ICG001 inhibits CRC cell growth and invasion, we explored the PI3K/AKT signal-

Figure 3. ICG001 induces caspase-dependent apoptosis by downregulating the expression of Bcl-2 and upregulat-ing the expression of Bad and Bax in HCT-116 cells. HCT-116 cells were exposed to ICG001 for 24 h, followed by western blotting with indicated antibodies. Means ± SEM. (n = 3). *P < 0.05, **P < 0.01.

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Figure 4. ICG001 inhibits the expression of eIF3D and its downstream PI3K/AKT signaling pathways in HCT-116 cells. Serumstarved HCT-116 cells were treated with ICG001 (0-2 mM) for 24 h, followed by stimulation with eIF3D (50 ng/ml) for 1 h. The cell lysates were subject to western blotting with indi-cated antibodies. Means ± SEM. (n = 3). ##P < 0.01, *P < 0.05, **P < 0.01.

ICG001 (0-2 mM) for 24 h, fol-lowed by stimulation with eIF3D (50 ng/ml). As indicat-ed in Figure 4, our western blot analysis showed that ICG001 inhibited the phos-phorylated PI3K and phos-phorylated AKT in a concen-tration dependent manner. Interestingly, ICG001 showed the most potent inhibitory effect on p-PI3K and p-AKT meanwhile the eIF3D expres-sion was shown to be lowest at 1 mM. Thus, our data fur-ther support that ICG001 in- hibited CRC cell growth and invasion via blockade of p- PI3K, p-AKT and eIF3D acti- vations.

ICG001 inhibits HCT-116 xe-nograft growth in nude mice

To evaluate the anti-tumour activity of ICG001 in vivo, HCT-116 xenograft model was used. In this model, HCT-116 cells were injected subcuta-neously into the hind flank of each nude mouse, then the mice were randomised into four groups (6 mice per gro- up) when the tumours grew to a size of about 100 mm3. Next, the animals were orally given three doses (12.5, 25, and 50 mg/kg) of ICG001 or vehicle (control) every day. At the end of the experiment, we found that ICG001 dose-dependently inhibited the tu- mour growth (volume) (Figure 5A) compared with the vehi-cle. Similarly, treatment with ICG001 also inhibited the tumour weight increase sig-nificantly, compared with the vehicle treatment (Figure 5B). Of note, no obvious toxicity was observed in all the treat-ed groups (Figure 5C).

Figure 5. In vivo anti-tumour activity and mechanism of action of ICG001. A-C. Nude mice bearing HCT-116 tumour cells were treated with ICG001 at the indicated doses or vehicle control alone over 28 days. The bodyweight of mice and tumor sizes were measured every three days and at the end of the experiments, the mice were killed and the tumour tissues were dissected and weighed. The data are expressed as the Mean ± SEM. of groups (6 mice per group). The representative images of isolated tumours are also shown. *P < 0.05; **P < 0.01.

ing in CRC cells by western blot. For this, se- rum-starved HCT-116 cells were treated with

To elucidate the mechanism of anti-tumour action of ICG001 in vivo, immunohistochemi-

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cal analysis was conducted using HCT-116 tu- mour. As shown in Figure 6, ICG001 showed anti-cancer active (HE staining) and a decre- ased tumour cell proliferation (Ki67-positive staining) and a significant increase in apopto- sis (LC3B-positive staining), compared with the vehicle. Moreover, ICG001 remarkably reduced the microvessel density (CD31 staining).

Discussion

The study provides the first evidence demon-strating the potent anticancer activity of ICG- 001 against CRC in vitro and in vivo, by target-ing eIF3D signaling. In vitro assays suggest that ICG001 inhibited cell proliferation and induced cell death in CRC cells. Furthermore, we were able to show that ICG001 induced apoptosis by flow cytometry. As shown in Figure 2, treatment with ICG001 for 72 h induced apoptosis in HCT-116 cells in a concentration-dependent man-ner, while our Western blot results suggest that ICG001 induces caspase-dependent apoptotic cell death by downregulating the expression of

the anti-apoptotic protein Bcl-2 and upre-gulat-ing the expression of the pro-apoptotic proteins Bad and Bax in CRC cells.

The PI3K/AKT signaling pathway has been extensively studied due to its critical role in cancer progression [14], including the regula-tion of metabolism, tumor growth, survival, and metastases [15]. Recently, therapeutic com-pounds that target this pathway have been developed and are currently being evaluated in clinical trials for several malignancies, includ-ing colon cancer [16]. Given recent evidence that the PI3K/AKT signaling pathway is over- activated in cancer cells [17, 18], several drugs that target PI3K and AKT are in development, a number of which have been validated in clinical trials. Subsequent addition of ionizing radiation can improve the anticancer effects of these drugs [19, 20].

The signaling events triggered by PI3K and AKT are complex, with different, partially overlap-ping functions that regulate cell survival and

Figure 6. Tumour tissues were evaluated by H&E, and immunohistochemistry with indicated antibodies. Represen-tative images are shown (200 ×).

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therapeutic resistance. Treatment with 1 mM ICG001 for 24 h inhibited the phosphorylation of PI3K and Akt significantly (Figure 4). There- fore, the more potent in vitro anticancer activity of ICG001 may be attributed to the fact that ICG001 exhibited stronger inhibition of eIF3D as opposed to its downstream effectors (PI3K and Akt). Our in vivo results show that at high doses (50 mg/kg) ICG001 could significantly inhibit HCT-116 xenograft growth when orally administered to mice (Figure 5), consistent with the effect seen in vitro. Further research will be necessary to evaluate the pharmacoki-netic profile of ICG001 in animals; such data would be instructive to help develop derivati- ves of ICG001 with improved pharmacokinetic properties.

Taken together, our data show that ICG001 inhibits proliferation and induced apoptosis in CRC cells in vitro and in vivo. ICG001 exerts its anticancer action, at least in part, by inhibiting eIF3D-mediated PI3K/AKT signaling pathways. Our findings suggest that ICG001 is a novel inhibitor of eIF3D with great potential for the treatment of CRC and other tumors.

Disclosure of conflict of interest

None.

Address Correspondence to: Chenggang Yang, De- partment of Gastrointestinal Surgery, Liaocheng People’s Hospital, Liaocheng 252000, Shandong, China. Tel: +86-635-8276110; Fax: +86635-827- 7306; E-mail: Bakerham123@163.com

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