1370
considered a target for overcoming acquired anticancer anticancer resistance genes [15]. The aryl hydrocarbon receptor (AHR) and aryl hydrocarbon nuclear translocator (ARNT) (also known as hypoxia- inducible factor (HIF)- 1β) are a member of the basic helix- loop- helix PER/AHR/ARNT/SIM (bHLH- PAS) family of transcription factors [16]. Under normoxic conditions, ARNT serves as a dimerization partner for several transcription factors and multidrug resistance 1 1 –Our laboratory constructed an apoptin- derived peptide (AdP) as an antitumor polypeptide that was originally designed based on the structure of the apoptosis hormone. Our previous study showed that AdP has anticancer activi-ties in vitro and in vivo by promoting apoptosis and inhibiting metastasis [21]. In addition, we found that AdP inhibits MMP- 9 expression through inactivation of PI3K/AKT/mTOR signaling [22]. However, no data regarding AdP and drug- resistant GC are available.Apoptin- derived peptide contains an SH3 domain, and it binds speci cally to PI3K, thereby inhibiting tumor cell growth. Our preliminary results show that AdP down-GC cells) and SW-620 (colon cancer cells) were obtained Hospital of Harbin Medical University. Human embryonic kidney (HEK) 293 cells were obtained from the Shanghai Co.Ltd, Zhejiang, China) and 1% penicillin–streptomycin solution (Beyotime Biotechnology, Shanghai, China). SGC-7901/CDDP was cultured with its maximum cisplatin actin (1:5000; Then, the membranes were incubated with secondary time PCRReagent (Invitrogen, Carlsbad, CA) and reverse-(Haigene, Shenzhen,China), following the manufacturer’s he human GC cell line SGC-7901 (cisplatin-
1371
assay (Amersco, Ohio). Cells at a density of 1~2620 with AdP at concentrations of at room temperature for 10min, and the optical density 7901 and SGC-7901/CDDP cells were cultured (Zhongshan Goldenbridge Biotechnology). Wax seal tissue slices were dewaxed, rehydrated, and then boiled in a ples were incubated with the secondary antibody at room no staining; 1+, minimum staining; 2+, moderate to strong staining of at least 20% of cells; 3+, strong staining of permeable nucleic acid stains, which are useful for the toring the emission spectral shifts of the dyes. Cells were unbound dyes. Observation and photography were perbrane integrity of normal cells and incorporate into the nuclear DNA to emit bright green uorescence. Bromine Bromium (EB) can only penetrate the damaged cells through the cell membrane, embedded in nuclear DNA to emit red uorescence. The cells treated with AdP h were digested by trypsin (Beyotime Biotechnology). The cell suspension was colµL of AO (Nachuan µg/mL), and µg/mL) was added to the suspension min. Samples were examined with a Live Cell Workstation (Leica AF6000,
Table Primer sequences of genes.
PI3K5-5-AKT5-5--5-5-
1372
tosis detection kit (Beyotime Biotechnology) according to well h, cells were washed three times with PBS and xed in 4 % min. Then, cells were stained with µg/mL) in PBS at room temperature min, and then washed to remove unbound dyes. ve were from patients with effective treatment by chemotwo courses of the same chemotherapy. The speci c inforin human GC PI3K/AKT protein expression in GC tissues and TCGA databases, which are available through cBioStatistical analysis of data was performed using SPSS version 13.0 for Windows (SPSS Inc, Chicago, IL). Data are presented as meanSE; Statistical comparisons among multiple were performed using ANOVA with a Holms–Sidak post hoc test and 0.05 was considered statistically signi cant.ni cant.structure of AdP is shown in Fig. 1.Inhibitory effects of AdP on viability of tumor cellsWe tested the effects of varying forms of AdP on the CDDP GC cells, 36.7µg/mL; MGC-803 cells, 44.43
Table Gastric cancer patient information.
VariablesGastric cancerGastric cancer resistanceNumber%Number%Age (years)59440330110220SexMale330330Female220220Classi cationHigh differentiated220550Medium differentiation33000Tumor typeAdenoma cancer550330Undifferentiated cancer00220Chemotherapy effectImprove55000No effect00550
Figure Diagram of the structure of AdP.
1373
excellent inhibitory effect on GC cells, and CDDP-showed that CDDP had no signi cant in uence on the [22]. In sharp contrast, AdP suppressed cell viability in observed chromatin condensation in SGC-7901 and SGC-Furthermore, we determined whether AdP treatment 7901 and SGC-7901/CDDP cells. When treated with 15forming ability of these two cell lines was substantially weakened (Fig.3C). CDDP (17901 cells
Figure Based on MTT experiments, the IC50 values of AdP were 407901), 36.77901/CDDP), 44.43(MGC-803), 50.7620), 80
1374
1375
To evaluate the effect of AdP on cell cycle arrest, we 7901/CDDP 7901cells treated with different concentrations h. The proportion of GC cells in the G2/M phase increased. These results suggest that AdP can increase the G2/M phase population, thereby leading to apoptosis, and the AdP-treated 7901/CDDP cells have an increased sensitivity to To determine the clinical relevance of the expression of the above proteins, we rst analyzed their expression in clinical specimens in the human protein atlas (www.proteinatlas.org). GC cells exhibited positive expression of AKT, MDR1, and ARNT proteins, and normal stomach tissues had negative or weak expression of these proteins. In particular, the expression of PI3K and ARNT in GC tissues was signi cantly higher than that in normal stomach tissues (Fig.4A). To verify whether elevated p-AKT, MDR1, and ARNT expression confer CDDP resistance in GC cells, we rst determined the protein levels of AKT, MDR1, and ARNT in GC and CDDP-resistant GC tissues relative to that in normal gastric epithelium. An immunohistochemistry (IHC) analysis was performed to measure the expression of p-AKT, MDR1, and ARNT in a series of wax-sealed human platinum-resistant GC samples (Fig.4B). The results revealed that the expression of these proteins in platinum-resistant GC tissues was higher than that in normal GC tissues. As shown in Fig.4C, at the cellular level, we found that p-p85, p85, AKT, AKT, ARNT, and MDR1 were highly expressed 7901/CDDP cells. However, the expression of these proteins was signi cantly down-regulated by AdP at a concentration 60µg/mL. Our results showed a reasonably good correlation between the expression of these proteins and CDDP resistance for most of the GC cell lines examined.human GC cells [24]. We found that SGC-tissue. AdP at concentrations from 40µg/mL to 100100-tions as an ATP- dependent drug ef ux pump that reduces the intracellular concentrations of chemotherapeutic agents, including CDDP [26]. Our results indicated that MDR1 expression was down-ing. Our results showed that ARNT expression was markmarkthat AdP might regulate ARNT protein levels by inhibit-ing the PI3K/AKT signaling pathway. To examine this notion, we used SCG-7901/CDDP cells treated with CDDP blotting results demonstrated that IGF-1 and AdP cotreat
the standard deviation of three independent h µg/mL). (C) Plate cloning experiment. The AdP concentration forming ability of the two cell lines reached the V and propidium iodide and then subjected to ow cytometry. The data are presented as the meanstandard deviation of three independent 0.05.(E) ADP leads to S phase reduction, and G2/M phase increased in SGC-7901 as same as SGC-7901/CDDP, thereby
1376
activator IGF-1 and AdP promoted SGC-7901/CDDP cell proliferation and signi cantly weakened apoptosis, and
Figure PI3K, p-p85, p-AKT, ARNT, and HSP70 are overexpressed in human gastric cancer (GC) specimens; p-AKT, MDR1 and ARNT are highly expressed in platinum-resistant GC tissues. (A) PI3K, AKT, ARNT, MDR1, and HSP70 expression in normal stomach tissue and GC specimens. Images were taken from the Human Protein Atlas (http://www.proteinatlas.org) online database. (B) p-AKT, MDR1, and ARNT expression in platinum-resistant GC tissues was determined by immunohistochemical analysis (nP0.05 versus the control. (C) The expression levels of p-p85, p85, p-AKT, AKT, ARNT, and MDR1 in SGC-7901, SGC-7901/CDDP cells and with AdP (607901/CDDP cells were determined by immunoblotting.
1377
7901/CDDP cells were treated with AdP for the concentration gradient, and p-p85, p85, p-AKT, AKT were determined by immunoblotting assay. (c),
1378
6D shows that the mRNA levels of p85 PCR, were not altered by AdP. We further found that the mRNA levels of PI3K and AKT were significantly altered in only 30 human clinical samples of 265 patients from the TCGA 6E). This finding suggests that GC is associated with a low level of PI3K and AKT mRNA
Figure Apoptin-derived peptide treatment correlated with ARNT by PI3K/AKT signaling pathway. (A) SGC-7901/CDDP cells were treated with AdP (60p85, p-AKT, ARNT expression was determined by immunoblotting assay; P0.05. (B, C) Plate cloning and AO/EB. SGC-7901/CDDP cells were treated with PBS, AdP (601 for 24workstation. (D) qRT-PCR. p85 and AKT expression was not markedly changed in the presence of the AdP compared with that in the control group. (E) PI3K, AKT mRNA expression in the TCGA gastric cancer RNAseq (IlluminaHiSeq; n
1379
7901/CDDP cells, and PBS was used as a positive control. Then, invasion and migration experiments were performed. The results showed that AdP signi cantly reduced the number of invasive cells and effectively inhibited the migration of GC cells. As 7, AdP signi cantly inhibited the migration 7901/CDDP dependent manner. Our results suggest dependent tumor cell apoptosis without affecting normal somatic somatic exerts a strong inhibitory effect on glioma cells, especially on the invasion and migration of these cells [23]. GC cells and glioma cells both have a similarly strong ability to invade and migrate, and the acquired drug resistance in GC cells is closely associated with the activation of the PI3K/AKT signaling pathway [22]. It is known that AdP suppresses glioma cell invasion and migration via suppressing PI3K/AKT signaling [22]. Therefore, we pro-posed that AdP might be effective in treating gastric cancer ated the data in support of our hypothesis. These data outcomes [29]. We analyzed clinical drug-resistant GC GC that the PI3K/AKT signaling pathway is closely related to GC resistance to CDDP.AdP has an SH3- binding domain that can bind RTK receptors to inhibit p85 phosphorylation, which in turn inhibits AKT phosphorylation (p-AKT). We initially envisaged that AdP containing the SH3 domain could inhibit AKT and suppressed their activation to inhibit the PI3K/7901 cells.is a major cause of chemotherapy failure[32]. Increased Increased 34]. We found that MDR1 was involved in resistance to CDDP in GC cells, and AdP down- regulated MDR1 expres-sion, thereby increasing the sensitivity of GC cells to CDDP. Strikingly, one study reported that AKT activity decreases the suppression of YB1 phosphorylation, reduc-ing MDR1 expression, which can promote CDDP sensitivity in GC cells [35]. Therefore, we have reason to speculate that by inhibiting the PI3K/AKT pathway, AdP further inhibits YB1 phosphorylation and reduces MDR1 expresscription factors, including MDR1, thereby contributing contributing of this study show that ARNT expression in CDDP- resistant GC cells and tissues was higher than that in GC cells ARNT expression, and this down-regulation was achieved
1380
µg/mL, 20µg/mL and 30µg/mL) or PBS for 48h, and the migrating cells were visualized by phase-contrast imaging. The results are expressed as the mean±SEM of at least three independent experiments. *0.05, **0.01 compared with the control group. (B) Cells were treated with AdP
1381
They can induce tumor cell cycle arrest, which induced apoptosis. We found that AdP can reduce the S phase of SGC- 7901/CDDP cells and inhibit cell proliferation. Thus, we hypothesized that AdP had a better effect on CDDP- resistant GC. The LRS domain has a β- corner secondary structure, which largely protects the spatial structure and activity of NLS1 and NLS2, and the LRS domain is rich in leucine. Our previous studies showed that the LRS domain interacts with AKT and can inhibit AKT phosphorylation. In conclusion, we previously designed a synthetic apoptin- derived peptide, which super-imposes the functions of NLS1, NLS2, and LRS domain, while maintaining the relative space and activity of the three domains continuously maintained by the LRS domain. Each structure of AdP has important signi cance for apoptosis. Therefore, it is a potential treatment for revers-ing GC resistance.Development of novel chemo- sensitive antitumor drugs for clinical application is of great importance [38]. Here, we evaluated the effects of AdP and present several novel features that suggest the therapeutic ef cacy of AdP in GC. First, AdP had a speci c cytotoxic effect on GC cells of p-p85 and p-AKT. Third, a survey of clinical samples and that AdP down-regulated the expression of these be a novel therapeutic agent for the clinical treatment of opment of new drugs for GC and presents evidence that
Figure Inhibitory effect of AdP on SGC-7901/CDDP cell PI3K/AKT signaling pathway.
1382
This article does not contain any studies with human All applicable international, national, and/or institutional ticipants were in accordance with the ethical standards of the institutional and/or national research committee and Kim, Y. I., I. J. Choi, and M. C. Kook. 2014. The status and incidence of metachronous gastric cancer after endoscopic Koizumi, W., H. Narahara, T. Hara, A. Takagane, T. Akiya, M. Takagi, etal. 2008. S–1 plus cisplatin versus S–1 alone for rst-line treatment of advanced gastric cancer (SPIRITS trial): a phase III trial. Lancet Oncol.
1383
22.Song, W., H. Zhao, Z. Cui, X. Ma, W. Zhang, D. Wang, etderived peptide that interacts with SH3 domains and inhibits 23.Zhang, L., H. Zhao, Z. Cui, Y. Lv, W. Zhang, X. Ma, etglioma growth. Oncotarget8:31119–31132.24.Wang, D., Y. Xin, Y. Tian, W. Li, D. Sun, and Y. Yang. 2017. Pseudolaric acid B inhibits gastric cancer cell metastasis in vitro and in haematogenous mediated apoptosis pathways. Exp. Cell Res.. 352:34–44.25.Chen, K. G., and B. I. Sikic. 2012. Molecular pathways: resistance. Clin. Cancer Res. 18:1863–1869.26.Tang, B., Y. Zhang, R. Liang, Z. Gao, D. Sun, and L. Wang. 2013. RNAi-mediated EZH2 depletion decreases MDR1 expression and sensitizes multidrug-resistant hepatocellular carcinoma cells to chemotherapy. Oncol. 27.Peng, X., D. Zhou, X. Wang, Z. Hu, Y. Yan, and J. Huang. 2016. Matrine suppresses proliferation and invasion of SGC7901 cells through inactivation of PI3K/28.Rollano Peñaloza, O. M., M. Lewandowska, J. Stetefeld, K. Ossysek, M. Madej, J. Bereta, etselective anticancer agents. Trends Mol. Med. 29.Chen, J., D. Xia, J. D. Luo, and P. Wang. 2009. tumour effects and inhibits the EGFR/PI3K/Akt 11:669–677.30.Winder, A., K. Unno, Y. Yu, J. Lurain, and J. J. Kim. 2017. The allosteric AKT inhibitor, MK2206, decreases tumor growth and invasion in patient derived xenografts of endometrial cancer. Cancer Biol. Ther. 18: 958–964.31.Ebrahimi, S., M. Hosseini, S. Shahidsales, M. Maftouh, Cancer. Curr. Med. Chem. 24:1321–1331.32.Gottesman, M. M., T. Fojo, and S. E. Bates. 2002. Multidrug resistance in cancer: role of ATP-dependent transporters. Nat. Rev. Cancer 2:48–58.33.Zhang, D., and D. Fan. 2007. Multidrug resistance in 7:1369–1378.34.Xu, H. W., L. Xu, J. H. Hao, C. Y. Qin, and H. Liu. 2010. Expression of P-glycoprotein and multidrug resistance-associated protein is associated with multidrug35.Mo, D., H. Fang, K. Niu, J. Liu, M. Wu, S. Li, et2016. Human helicase RECQL4 drives cisplatin resistance in gastric cancer by activating an AKT-YB1-MDR1 signaling pathway. Cancer Res. 76:3057–3066.36.Jangamreddy, J. R., S. Panigrahi, K. Lot , M. Yadav, S. interaction with BCR-ABL1, and development ofbased targetedtherapy. Oncotarget 5:7198–7211.37.Zhang, L., J. Yang, L. Liu, W. Tian, M. Gao, W. Song, ability and tumor-killing activity of the combinatorial function domain of apoptin. Anticancer Drugs 28:401–409.38.Wu, J., H. Qin, T. Li, K. Cheng, J. Dong, M. Tian, etspeci c glycosylation of secreted proteins associated with multi-drug resistance of gastric cancer. Oncotarget 7:25315–25327.
1369
Introduction
Gastric cancer (GC) is the third major cause of global
cancer-
related death [1, 2]. Because of its high incidence
and high mortality, GC is a serious threat to humans
[3, 4], and China is among the nations with the highest
incidence of GC [5]. The most frequently used chemo
-
therapeutic agent for GC treatment is cisplatin (CDDP)
[6]. Based on phase III trials in Japan, a combination of
CDDP and the 5-
uorouracil-
related drug S-
1 has been
considered the rst-
line chemotherapy treatment for
advanced GC [7]. However, the overall ef cacy of CDDP
treatment is limited in the clinic due to the development
of drug resistance [8]. Therefore, identifying new targets
involved in drug resistance may foster the development
of new strategies for improving chemotherapy targeting
GC.
The molecular mechanisms of drug resistance are com
-
plex and involve antiapoptosis [9, 10], drug metabolism,
and drug ef ux mechanisms [11]. One of the main mecha
-
nisms of CDDP resistance is the escape of tumor cells
from apoptosis [12, 13]. The PI3K/AKT pathway has been
ORIGINAL RESEARCH
Apoptin-
derived peptide reverses cisplatin resistance in
gastric cancer through the PI3K