Gallic acid enhances the antitumor activity of Icotinib hydrochloride in non-small cell lung cancer via of Hippo- YAP signaling pathway in vitro and in vivo

Dapeng Wang (  wangdapeng1031@163.com ) Chinese PLA General Hospital https://orcid.org/0000-0001-6193-098X Yinghua Guo Chinese PLA General Hospital Yuan Liu Chinese PLA General Hospital Rengerile Sa Inner Mongolia Medical University Junfeng Wang Chinese PLA General Hospital Bin Zhang Binzhou Medical College: Binzhou Medical University Po Bai Chinese PLA General Hospital Xiaolei Su Chinese PLA General Hospital Xuelin Zhang Chinese PLA General Hospital Yongqing Zhang Chinese PLA General Hospital Changting Liu Chinese PLA General Hospital


Introduction
As one of the most usual malignant tumors, lung cancer has been the malignant tumor with the greatest morbidity and mortality across the world since the 1990s [1]. Though the techniques of surgery and chemotherapy have been improved, the 5-year survival rate of patients is below 20% due to the limitations of surgical treatment and the side effects of chemotherapy drugs [2]. Tyrosine kinase inhibitors (TKIs) of epidermal growth factor receptor (EGFR), as the main driver gene in NSCLC has become one of research hotspots. The anticancer e cacies of current therapies are limited, due to the high degree of cancer clonal heterogeneity. In addition, using a single therapeutic agent is not effective in eradicating cancer cells, and hence the use of combinatorial therapy, which could inhibit multiple targets or redundant pathways simultaneously, is essential and inevitable. Scholars have evaluated a lot of methods such as integration of EGFR-TKIs with natural plant extract for progression delaying.
Gallic acid (GA, Figure1) is rich in grapes, tea, nuts and red wine as a plant extract and natural phenolic compound [3]. According to report, it shows various pharmacological and biological nature such as antibacterial activity [4], antiviral activity [5] and antioxidants [6]. The anti-tumor function of GA has been brought into focus recently.
According to researches, GA may stop the development of malignant tumors by stopping the propagation and aggressive activity of tumors [7][8][9][10][11]. Also, GA exerts an excellent inhibitory effect on the tumors such as cervical cancer [12], breast cancer [11], lung cancer [13] and prostate cancer [14]. Thus, it was expected that GA integrated with TKIs may play a better prohibitive role in NSCLC.
Icotinib hydrochloride (IH, Figure1) belongs to highly e cient and speci c EGFR-TKIs and was independently developed in China. Clinically, IH has been extensively applied in China with similar clinical potency and greater security compared with ge tinib [15][16]. The Hippo-YAP signaling pathway is able to adjust the organ size and maintain the dynamic balance between cell propagation and apoptosis as a signi cant signaling pathway and a tumor suppressor. Many studied have illustrated that the Hippo-YAP signaling pathway can participate in the development of tumor by affecting cell proliferation and apoptosis. YAP and caspase-3 are two important genes in Hippo-signaling pathway. However, whether YAP and caspase-3 are involved in GA-IH interaction has not been reported. This research aimed at evaluating the antitumor role of GA integrated with IH ,to explore the mechanisms underlying synergistic interactions between GA and IH, and provided a new therapeutic target for increased IH sensitivity. to 68 years old, with an average age of (59.24 ±5.76) years. Histological type was adenocarcinoma; All selected patients underwent radical resection without chemotherapy or radiotherapy before operation. The study was approved by the hospital ethics committee.

Cell Culture
Human NSCLC A549 and PC9 cell lines were cultured in RPMI-1640 medium containing 10% FBS at 37°Cin a humidi ed 5% CO 2 incubator. The medium was changed 2~3 times every week and the cells were used in their logarithmic growth phase.

Human lung tissue immunohistochemistry
Tissue specimens were xed in 10% neutral formalin solution for 48h, embedded in conventional para n. 3 ra n in convent were baked in 60°C oven for 30min, followed by dewaxing, antigen retrieval, antigen exposure, sealing and dripping peroxide. The operation steps were carried out according to the operating instructions of the Roche automatic immunohistochemistry instrument. The primary antibody dripped manually, and nally, it was counterstained with arti cial hematoxylin, blue back, and sealed with a neutral gum sheet. The primary antibody of immunohistochemistry was rabbit anti-human caspase-3, YAP, p-YAP polyclonal antibody (1:200, Santa Cruz, USA).
Immunohistochemical staining scoring was judged by the intensity of nuclear staining or cytoplasm staining in tumor tissues and paracancers. The widely accepted German semi-quantitative scoring system was used to score staining intensity and extent in different areas. Each specimen was assigned a score according to the intensity of the nuclei, cytoplasmic, and membrane staining (no staining¼0; weak staining¼1, moderatestaining¼2, and strong staining¼3) and the extent of stained cells (0-5%¼0,5-25%¼1, 26-50%¼2, 51-75%¼3 and 76-100%¼4). The nal immunoreactivity score was determined by multiplying the intensity score by the score for the extent of stained cells, generating a score that ranged from 0 (the minimumscore) to 12 (the maximum score).

Cell viability assay
According to the manufacturer's instructions, cell viability was evaluated using MTT (Solarbio, Beijing,China). Brie y, cells were seeded into 96-well plates at 8×10 3 cells per well and cultured for 48 hours (control group, GA group, IH Group, GA+IH group). Ten microlitres of MTT (concentration: 5mg/ml) solution was added into the medium in each well. After a 4 hr incubation, the supernatants were removed from each well, and 100μl DMSO was added to each well. The plate was incubated with shaking at room temperature for 15 minutes and red by a microplate reader (Bio-Tek Company,Winooski, VT) at a wavelength of 570nm. Each timepoint was repeated in three wells, and the experiment was independently performed three times.

Drug combination research
To evaluate the combined index (CI) of the effect of GA with IH on the growth of A549 and PC9 cells, the inhibitory effect of varying concentration of IH combined with varying concentrations of GA on the growth of A549 and PC9 cells was analyzed. CalcuSyn software (version2.0) was used to calculate the CI of GA combined with IH. CI > 1.1: antagonism; 0.9 CI 1.1: anadditive effect; 0.7< CI < 0.9: low synergy; 0.3 CI 0.7: synergy; 0.1 CI 0.3: strong synergy.

Cell apoptosis assay
Cell apoptosis was evaluated by ow cytometry using an Annexin V-FITC Apoptosis Detection Kit (KeyGen Biotech, Nanjing, China). Brie y, cells were seeded into 24-wellplates at 5×10 5 cells per well and cultured for 48 hr (control group, GA group, IH Group, GA+IH group). Then the cells were detached by trypsinization, washed twice in PBS (2000 r.p.m., 5 min), and resuspended in 500µl binding buffer. A volume of 5μl Annexin V-FITC and 10µl propidium iodide was added and mixed gently, and the cells were stained in the dark for 10 min at room temperature. The cells were analyzed immediately by ow cytometry (BD FACSCalibur, BD. Bioscience, San Diego, CA) and analyzed using FLOWJO software (FlowJo, Ashland, OR). The experiment was repeated three times.

Transwell migration assay
The ability of cell migration was measured by Transwell assay. The cells density was adjusted to 5×10 5 cells/ml. In addition, 200μL cell suspension was put into the upper chamber of Transwell cells, and then 600μL complete medium (control group, GA group, IH Group, GA+IH group) was added to the lower chamber of 24-well culture plate.
The culture plate was placed in a CO 2 incubator at 37°C for 48 hr. It was took out the Transwell, and washed twice with PBS. In addition, the matrix glue and the cells in the chamber were wiped off with a cotton swab. Moreover, it was soaked in 70% methanol for 30min, and dyed with crystal violet. After crystal violet staining, the stained migrating cells can be decolorized with 33% acetic acid, crystal violet can be completely eluted, and then the OD value of the eluent at 570nm was determined by microplate reader, which indirectly re ected the number of migrating cells. The experiment was repeated at least three times.

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A549 and PC9 cells, passaged in a 6-well plate at 2×10 5 /L, were cultured in an incubator for 24 hr, and different intervention drugs were added (control group, GA group, IH Group, GA+IH group). After 48 hr of treatment, cells were collected, total RNA was extracted with RNA Isolation Kit (SinoGene, Beijing, Chaina) and reverse transcribed using The First Strand Synthsis Kit (SinoGene, Beijing, China). The qPCR was performed using SYBR MasterMix Kit (SinoGene, Beijing, China). ACTB was used as an internal reference gene to detect the expression of Caspase-3 and YAP (Primers in the Table 1).The2 -△△Ct method was used to caclulate the relative mRNA expression.

Western blot
Cells were treated with GA, IH, and GA+IH for 48 hours and then harvested and lysed in RIPA buffer (SinoGene, China) containing 1% phosphatase for total protein extraction. 30 µg proteins per samples were separated via SDS-PAGE and transferred to PVDF membranes after quanti cation by the Pierce BCA protein assay Kit (Thermo Fsiher Sceinetifc, USA) and boiled for 10 minutes with protein loading buffer. Protein Marker (SM26616, Thermo Fermentas, USA) was loaded at the rst lane. the membranes were incubated with different primary antibodies anticaspase-3, YAP, p-YAP (1:1000) at room temperature for 1 hour and then exposed to HRP-conjugated secondary antibody (1:3000) for 1 hr. Finally, membranes were exposed to lms with ECL luminescent solution (29050, Engreen, China) in the darkroom. Beta-actin (1:3000, AC028, Abclonal, China) was as loading control.

Animal study
A total of 24 BALB/C nude mice (4 weeks old, 18-22 g) were purchased from the Experimental Animal Center. The study was approved by the Research Ethics Committee oftheChinease PLA General Hospital. Under sterile conditions, 0.2 ml (PC9) of cell suspension (l×10 7 cells/mL) was pipetted with a 1 mL disposable syringe and slowly injected into the right armpit of the nude mice. The long diameter of the tumor block >1 mm and felted lumps was indicative of the successful establishment of the NSCLC mouse model. After successful modeling, animals were divied into 4 groups: control group, GA group (30μg/kg), IH group (100mg/kg), and combined treatment group (30μg/kg+100mg/kg) and thenadministration for 14 d (1 time/d). During the administration, the long diameter and short diameter of the transplanted tumors were measured by vernier calipers every 3d, repeated in triplicate to obtain the average values. The tumor volume was then calculated: V=long diameter × short diameter 2 /2, and the growth curve of the transplanted tumors were plotted based on the values obtained. Each mouse was heparinized by intraperitoneal injection of 0.5% heparin (0.5mL), and then eye blood were collected to measure the levels of alanine aminotransferase (ALT) and alkaline phosphatase (ALP), blood urea nitrogen (BUN) and creatinine (Cr) after the last administration on the 14th day. In addition, the animals were euthanized with carbon dioxide asphyxiation, the tumors were removed, and the volumes of the tumors were measured after weighing the animals. In addition, protein expressions of caspase-3, YAP and p-YAP in tumors was detected by immunohistochemical staining, and the percentage of caspase-3, YAP and p-YAP protein positive staining was quanti ed. A microscope (IX51Olympus, Japan) and Image Pro Plus software were used to take photos and analyze slides.

Statistical analysis
The quantitative experiments were performed in triplicates, and the data were represented as the mean ± standard deviation (S.D.). A statistical comparison of the data was conducted using One-way ANOVA followed by Tukey's honest signi cant difference (HSD) test in post-hoc comparisons. Statistical signi cance between two groups was de ned as P <0.05.

Immunohistochemical staining of human NSCLC and paired paracancers
First of all, the expression of caspase-3, YAP and p-YAP in 6 NSCLC specimens were analyzed by IHC. The results showed caspase-3 was high expressed in normal lung tissues, but weakly expressed in NSCLC tissues. Compared with normal lung tissues, more YAP expression was detected in NSCLC tissues; Otherwise, the phosphorylation of YAP was downregulated in NSCLC tissues. In immunohistochemical staining, the positive granules were yellow or brown. All of them were located in the nucleus, and were granular, dispersed and/or mixed (Figure 2 ; Table 3B). Therefore, we used non-cytotoxic 0.5, 1.0, 5.0µM IH in combination with 66 and 132µM GA to evaluate the sensitization of GA to IH.
3.3 GA enhanced the drug susceptibility of IH in A549/PC9 Cells A549/PC9 cells were inoculated into 96 well plates with 4×10 3 cells per well, the cells were treated with IH (0.5, 1.0, 5.0µM) in combination with GA (66 and 132µM), and the cell survival rate was measured by MTT Assay. The synergistic antitumor effect of IH and GA was evaluated using COMPUSYN software. The Synergy Index (CI) was used to evaluate the synergy between GA and IH. Combinated 0.5,1.0,5.0µM IH and 66,132µM GA showed a abvious synergism ( Figure 3C-D). Thus, IH and GA yield a synergistic effect in killing A549/PC9 cells. Speci cally, 132µM GA was stronger than that treated with 66µM GA, 132µM GA did not yield measurable impacton cell viability of A549/PC9 cells, but clearly enhanced the sensitivity of A549/PC9 to IH. Also, as shown in Figure 3C, the e ciency of 5µM (A549) or 0.5µM (PC9) IH combined with 132 µM GA peaked at 48h. Here 132µM GA and 5µM(A549) or 0.5µM (PC9) IH have synergistic index of 0.65 and 0.67 (Table 6). Thus, IH And GA yielda synergistic effect in killing A549/PC9 cells. So 132µM GA and 5µM (A549) or 0.5µM (PC9) IH were used for further study.

GA Increased IH-induced apoptosis
We examined whether GA could enhance IH-induced apoptosis using ow cytometry in A549/PC9 cells. Apoptosis plays a key role in governing anticancer therapy, yet its involved mechanisms are largely unknown. As observed, the number of apoptosis were elevated in A549/PC9 cells exposed to co-treatment of IH and GA, compared with IH alone, or GA alone (Figure 4), indicating GA indeed enhanced IH-induced apoptosis. The results showed that the apoptosis rate in the combination group (30.93%/33.54%) was signi cantly higher than that in the GA (17.59%/17.55%) and IH (27.31%/32.32%) groups in the A549/PC9 cell groups (Figure 4, *P<0.05 **P<0.01 ***P<0.001). Together, these ndings suggested that GA may promote IH-induced apoptosis, of which was not cytoprotective, but lead to cell death.

GA enhances IH suppressed migration
It was detected by Transwell test in order to study the effect of GA, IH and combination therapy on the migration of A549 and PC9 cells. The absorbance of acetic acid solution was read by microplate reader (570nm). The results showed signi cant inhibitory effects on the migration of A549 and PC9 cells were in the three treatment groups.
Furthermore, the inhibition of migration of lung cancer cells was more obvious in the combination groups compared with the single drug group ( Figure 5).

GA enhances IH on mRNA expression of the key gene
The mRNA expression of Caspase-3 and YAP in A549 and PC9 cells were analyzed by qPCR. As a result, compared with the control group, the mRNA expressions of Caspase-3 were increased and YAP were downregulated in both the single drug group and the combination group, especially in the combination group ( Figure 6).

GA Enhanced IH-Induced caspase-3-dependent apoptosis through suppressing
Hippo-YAP signaling pathway Similarly, we determine whether Hippo-YAP pathway participated in GA and IH-induced apoptosis. Caspase-3 is activated during apoptosis. Given that caspase-3 plays a distinct role in IH-mediated apoptosis in cancer cells (Li et al., 2015). Combination of IH and GA signi cantly increased caspase-3 when compared with IH alone. we examined caspase-3 expression by western blots. In the presence of GA, the IH-induced expression of caspase-3 was markedly enhanced ( Figure 7A). Western blot analysis that the high expression of caspase-3 induced by the combination treatment in A549/PC9 cells (Figure 7). The expression level of YAP in the treatment group was signi cantly lower than that in the control group (Figure 7). This suggested that DNA repair may be blocked and induce apoptosis.
These results suggested that p-YAP may be involved in regulating the anti-tumor effect of GA on A549 and PC9; GA may promote apoptosis through suppression the expression of YAP and enhance the anti-tumor effect of IH. Thus, GA enhanced IH-induced caspase-3-dependent apoptosis through suppressed the Hippo-YAP signaling pathway.

GA enhanced antitumor e cacy of IH in vivo
To verify our previous conclusions, we established xenograft mouse model. The PC9 cells were subcutaneously implanted into BALB/c nude mice, and the mice were treated with IH with or without GA. As we have shown IH signi cantly inhibited tumor growth, and this e ciency was signi cantly enhanced by GA treatment (Figure 8a-b; Table7). Furthermore, due to the toxicity of IH, the on liver and kidney function of mice treated by IH was increased.
However, in the presence of GA, the reaction was recovered (Table 7). Moreover, the combination of IH with GA increased the expression of caspase-3, P-YAP and reduced compared with IH alone (Figure 8c). Collectively, GA could notably improve anti-tumor effect of IH, and reduce the toxicity generated by IH in vivo.

Discussion
IH, one of the most commonly targeted Drugs, has been widely used to treat NSCLC [17]. However, its tumoricidal e cacy is often limited. Multiple mechanisms contributed to IH sensitivity have been documented, such as decreased drug absorption and inactivated apoptosis programs [18]. Unfortunately, there remains a lack of targeted treatment strategies focusing on enhance IH sensitivity. To evade this di culty, we focused on looking for novel drugs to IH sensitivity. Our results offer the evidence that GA improved the sensitivity of A549/PC9 to IH. In addition, our study shows that GA may potentiate antitumor activity of IH in NSCLC line A549/PC9, suggesting the synergism of GA and IH was applied to NSCLC cells As everyone knows, two hallmarks of cancer are proliferation sustaining and apoptosis inhibiting [19]. Proliferation can be sustained by cancer cells through the production of development elements themselves or stimulation of normal cells to offer different development elements [20][21]. Apoptosis is the predominant manner of IH-induced cell death. However, tumor cells may develop its own speci c strategies to evade apoptosis, which facilitates their survival and promote resistance to anticancer therapies. Caspase-3 is a apoptotic effector that has been well studied to be involved in IH-induced apoptosis. In this study, IH markedly induced caspase-3-dependent apoptosis, of note, its e ciency was enhanced by GA.
Hippo exerts a great role in tumor development and progression [22][23]. YAP is a core signaling pathway participating in Hippo [24][25], involved in Hippo pathway that is hyperactivated in a high many types of tumors, which drives cancer growth, inhibits apoptosis, promotes invasion and angiogenesis [26]. IH restrain Hippo activation is an key regulator of the YAP response to apoptosis caused by IH. Our data show that GA serves an antiangiogenesis inducer that potentiated IH-induced p-YAP activation,suppressed YAP expression. Thus, cotreatment of IH and GA induced caspase-3-mediated apoptosis via suppressing Hippo-YAP pathway.
It is well established that Hippo-YAP pathway is emerging as important player in various types of cancers [27]. For example, in prostate cancer, inhibition of the Hippo-YAP pathway is su cient to activate apoptosis [28]. Indeed, cotreatment of IH and GA signi cantly increased the level of phosphorylated YAP in A549/PC9 cells. The YAP oncogene has been identi ed as a potential target for cancer therapy. Activation of YAP contributes to the proliferation and invasion of cancer cells, and has been also considered as a biomarker in several tumor types [29].
Of note, YAP is regarded as a downstream factor of the Hippo pathway [30]. Here, we found that IH or GA alone could suppressed YAP level, however, the e ciency of combination treatment was stronger. These ndings clearly indicated that GA enhanced IH-induced apoptosis via suppressing the Hippo-YAP signaling pathway. Furthermore, our in vivo experiment indicated that GA signi cantly enhanced IH-suppressed tumor growth, and reduced the toxicity generated by IH.
In sum, our results showed that GA enhances the antitumor activity of IH in NSCLC cells. Mechanically, GA enhanced IH-induced apoptosis via Hippo-YAP signaling pathways, suggesting that Hippo-YAP signaling was the direct target of GA ( Figure 10). Notably, GA may reduce the toxicity associated with repeated administration of IH in tumorbearing mice. These ndings suggest that the combination of IH treatment with GA may be effectively applied for the treatment of NSCLC. In this study, A549/PC9 cells are a relatively homogeneous cell type, it is not clear whether the combination therapy is effective to other lung cancer cells. Thus, further experiments should performed to demonstrate whether GA may enhance the sensitivity of other lung cancer cells to IH.

Declaration of Con icting Interests
The author(s) declared no potential con icts of interest with respect to the research, authorship, and/or publication of this article.

Ethical statement
The current study was performed with the approval of the Ethics Committee of the the Chinese PLA General Hospital. This work was in conformity with the principles of the Helsinki Declaration. All the participants voluntarily  Tables   Table 1 Primers for the target genes used in qPCR Gene Name Size bp Primers Sequences (5′ to 3′)      Table 8 Protective effect of GA on IH-induced liver and nephrotoxicity   GA enhanced IH-induced apoptosis. Cells were exposed to IH with or without GA for 48 h, cell apoptosis was measured by ow cytometric analysis.
(C) Western blot analysis showing caspase-3, cleaved caspase-3 and PARP expression levels in A549/DDP cells treated as indicated. Actin was used as loading control. Data are representative of three independent experiments (mean ± SEM).

Figure 5
GA enhances IH suppressed migration. Cells were exposed to IH with or without GA for 48 h, cell apoptosis was measured by crystal violet (a: A549; b: PC9). Data are representative of three independent experiments (mean ± SEM).    A mechanism map depicting the role of GA combinated with IH in the progression of NSCLC via YAP-Caspase-3 axis.
Gallic acid combinated with IH disrupts the activation of YAP to enhance the expression of Caspase-3, whereby inhibiting the carcinogenic activity of YAP and repressing the development of NSCLC.