Therapeutic Mechanism of Lapatinib Combined with Sulforaphane on Gastric Cancer

Background Lapatinib is a small-molecule tyrosine kinase inhibitor that plays important roles in cell proliferation and survival. Administration of lapatinib with capecitabine is an effective treatment for HER2-positive metastatic BC. However, the effects of lapatinib on gastric cancer (GC) remain to be clear. In this study, we aimed to investigate the therapeutic effects of lapatinib combined with sulforaphane on GC and its underlying mechanisms. Methods SGC-7901 and lapatinib-resistant SGC-7901 cells were treated with lapatinib (0.2 μM), sulforaphane (5 μM), or their combinations. Cell viability, invasion, cycle, and apoptosis of SGC-7901 and lapatinib-resistant SGC-7901 cells were evaluated by thiazolyl blue tetrazolium bromide (MTT), Boyden chamber assay, and flow cytometer. The protein expressions of HER-2, p-HER-2, AKT, p-AKT, ERK, and p-ERK were detected by Western blotting. Results We observed that lapatinib combined with sulforaphane significantly decreased cell viability and inhibited cell migration of drug-sensitive and drug-resistant cells. Lapatinib sulforaphane also remarkably induced cell apoptosis with G0/G1 arrest. In addition, Western blotting revealed that the expressions of HER-2, p-HER-2, AKT, p-AKT, ERK, and p-ERK were downregulated by lapatinib-sulforaphane treatment. Conclusion Combination of lapatinib and sulforaphane might be a novel and promising therapeutic treatment for lapatinib-sensitive or lapatinib-resistant GC patients.


Introduction
Gastric cancer (GC) is one of the most common malignant tumors worldwide, with high morbidity and mortality [1]. In 2018, there were 1,033,701 new cases of GC and 782,685 GC related deaths all over the world [1]. Moreover, 60% of world total cases and the highest incidence rates occur in Eastern Asia [1]. Although the diagnosis and treatment methods of GC have greatly improved, the five-year survival rate of GC patient is still 10-30% [2]. Development and progression of GC are regulated by many environmental and genetic risk factors, including Helicobacter pylori (H. pylori) infection, unhealthy dietary habits, alcohol consumption, smoking, obesity, gastric diseases, and others [3]. e routine treatments of GC include surgery, chemotherapy, and radiotherapy. Recently, molecular targeted treatment for GC has gradually deepened, which has greatly improved the efficiency of GC treatment and played an important role in the individualized treatment of GC [4,5].

Up To.
Lapatinib is the first dual inhibitor of epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2) tyrosine kinases, which reversibly binds to the cytoplasmic ATP-binding site of the kinase domain and blocks signal transduction cascade from the receptor [6]. is interaction leads to decreased growth, metastasis and angiogenesis, increased apoptosis, and genetic instability by affecting the Ras/Raf mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3 kinase (PI3-K)/AKT signaling pathway [7]. Recently, increasing evidence indicated that HER2 might play an important role in the development and progression of GC [8]. Several preclinical studies have reported the antitumor activity of lapatinib in HER2-positive GC cell lines, suggesting the therapeutic effects of lapatinib for GC [9]. In a randomized placebo-controlled phase II study, lapatinib with epirubicin, cisplatin, and 5-fluorouracil or capecitabine was effective in GC patients, but not to patients with metastatic GC [10]. In addition, primary or acquired resistance to lapatinib still occurs in cancer therapy [11].
Sulforaphane is a natural isothiocyanate derived from cruciferous plants, such as broccoli, brussels sprouts, and cabbage. Sulforaphane has antioxidant and anticancer properties by triggering cell cycle arrest, apoptosis, and angiogenesis prevention [12]. In pancreatic cancer cells, sulforaphane inhibits sonic hedgehog (SHH) and PI3/ AKT signaling pathway by reducing NF-kB's DNA binding capacity [13]. Furthermore, Mondal et al. reported that sulforaphane induced apoptosis and inhibited migration of human GC cells [14]. In vivo, studies presented that sulforaphane suppressed azoxymethane-induced colonic aberrant crypt foci (ACF) and prevented polyps in Apc/Min mice [15,16]. Importantly, sulforaphane combined with other anticancer compounds could be more effective to target multiple pathways involved in cancer. For example, sulforaphane enhanced the efficacy of imatinib and sorafenib in pancreatic cancer cells and chronic myeloid leukemia cells, individually [17]. In HER2-positive breast cancer (BC) cells, sulforaphane not only potentiated antiproliferative activity of lapatinib but also was related to more effective induction of apoptosis [18]. However, the efficacy of combination therapy involving lapatinib and sulforaphane in GC remains unknown. erefore, we hypothesized that lapatinib combined with sulforaphane might be more effective in the treatment of GC. In this study, we investigated the molecular mechanisms associated with the synergistic antitumor effects observed following treatment with lapatinib and sulforaphane, as well as the efficacy of interactions between lapatinib and sulforaphane in SGC-7901 and lapatinib-resistant SGC-7901 cells.

Apoptosis
Assay. Annexin V-FITC/PI apoptosis detection kit (Nanjing Jiancheng, China) was used to measure the apoptosis of SGC-7901 and lapatinib-resistant SGC-7901 cells in a similar procedure as above but without fixing in 70% ethanol. e cells were washed with PBS and resuspended in 1x binding buffer. Next, Annexin V-FITC and PI were added to stain for 15 min. e apoptosis cells were measured using the flow cytometer (BD, USA).

Western
Blotting. SGC-7901 and lapatinib-resistant SGC-7901 cells were seeded in 4 ml of medium in 6 cm plate for 24 h, and the medium was replaced with lapatinib (0.2 μM), sulforaphane (5 μM), or combinations of compounds. After 48 h, total proteins were extracted with RIPA lysis buffer (Beyotime Biotechnology, China). e samples were loaded and separated by 12% sodium dodecyl sulfatepolyacrylamide gel electrophoresis and then transferred onto nitrocellulose membranes. e membranes were washed with PBS and blocked in 5% skim milk, followed by immunoblotted using the following primary antibodies: HER-2, p-HER-2, AKT, p-AKT, ERK, p-ERK, and β-actin overnight at 4°C. e anti-mouse and anti-rabbit antibodies conjugated with HRP were used for incubation. e proteins were visualized using the enhanced chemiluminescence (ECL) kit. Finally, the intensity of Western blot bands was analyzed with Quantity One 1D Analysis Software (Bio-Rad, USA).

Statistical Analysis.
e results were presented as the mean ± standard deviation and analyzed by SPSS 16.0 software (IBM, USA). We performed the graphics using GraphPad Prism software (USA). Differences between groups in invasion tests were analyzed with Student's t test. P < 0.05 was considered as significantly different.

e Effects of Lapatinib, Sulforaphane, and eir Combination on Viability of Drug-Sensitive and Drug-Resistant SGC-7901 Cells.
To determine the influence of combined treatment on viability of the two cell lines, we performed the MTT assay. According to previous studies, we treated SGC-7901 and lapatinib-resistant SGC-7901 with 0.2 μM sulforaphane, 5 μM lapatinib, or their combinations for 48 h. As shown in Figure 1, lapatinib was almost as effective as sulforaphane in populations with low percentage of resistant cells, and combined therapy was the most effective. When percentage of lapatinib-resistant cells was more than >25%, lapatinib alone had the lowest efficacy. However, the low viability level was maintained due to sulforaphane activity, either alone or in combination with lapatinib.

Inhibition of Invasion of the Drug-Sensitive and Drug-Resistant SGC-7901 Cells by the Combination of Lapatinib and Sulforaphane.
To investigate whether the combined treatment affects cell invasion, we conducted Boyden chamber assay. In Figure 2, cells were treated with lapatinib, sulforaphane, or their combination, the migrations of SGC-7901 and lapatinib-resistant SGC-7901 cells were significantly inhibited compared with control group (P < 0.05). In lapatinib-resistant SGC-7901 cell, although sulforaphane alone had no significant effect on cell invasion, combination of lapatinib with sulforaphane was observed to significantly inhibit cell invasion. Moreover, combined treatment most efficiently decreased invasion of SGC-7901 (P < 0.001) and lapatinib-resistant SGC-7901 (P < 0.001) cells.

Lapatinib-Sulforaphane Treatment Downregulated the
Expressions of HER-2, p-HER-2, AKT, p-AKT, ERK, and p-ERK. To investigate the possible mechanism underlying the lapatinib-sulforaphane treatment, we detected the expression of genes related to HER2 signaling pathway by Western blotting (Figures 5(a) and 5(b)). In SGC-7901 cells, we observed that lapatinib significantly inhibited the expressions of p-HER-2, p-AKT, and p-ERK (P < 0.05), but no remarkable effects on the expressions of HER-2, AKT, and ERK. In addition, HER-2, p-HER-2, AKT, p-AKT, ERK, and p-ERK expressions were significantly lower in sulforaphane and lapatinib-sulforaphane groups than those in the controls (P < 0.05). As shown in Figure 5(b), the expressions of HER-2, p-HER-2, AKT, p-AKT, ERK, and p-ERK were significantly downregulated in populations enriched in lapatinibresistant SGC-7901 cells. In SGC-7901 and lapatinib-resistant SGC-7901 cells, the inhibiting effect of lapatinib-sulforaphane treatment on the expressions of p-HER-2, p-AKT, and p-ERK were more significant than drug alone.

Discussion
In a large cohort of patients with GC, HER2 overexpression was reported as 11.8% of GC and was significantly related to aging, intestinal histology, clinical stages, venous invasion, and frequent lymph node metastasis [19]. Lapatinib is small molecule inhibitors of HER2, approved by the United States Food and Drug Administration for the treatment of HER2positive BC patients. Lapatinib also has clinical activity in women with advanced HER2-positive GC [20]. However, primary or acquired resistance against treatment with HER2   Evidence-Based Complementary and Alternative Medicine 5 inhibitor is an unresolved issue in clinical oncology. Several molecular mechanisms underlying the resistance to lapatinib have been proposed, including mutations within genes of HER2 or EGFR receptors, abnormal expression of hormones, hyperactivation of PIK3Ca, or mutations in PI3K pathway gene [11,21]. Chen et al. indicated that activation of receptor tyrosine kinases promotes resistance to lapatinib in patients with HER2-positive GC [20]. e combination of phytochemicals with lapatinib is the potential way to overcome drug resistance. Phytochemicals, including sulforaphane, are important tools of cancer prevention and therapy [22]. e anticancer efficacy of sulforaphane was reported to be associated with ROS production, mitosis-specific arrest during cell cycle, and blocking of several signaling pathway [23,24]. To address the question of whether lapatinib-sulforaphane treatment might be effective for GC and especially lapatinib-resistant GC, we firstly cultured cell-based model of lapatinib resistance using SGC-7901 cells. We further mixed SGC-7901 and lapatinib-resistant SGC-7901 cells in various proportions, which might mimic heterogeneity of gastric tumor in context of the resistance acquisition process. MTT assay revealed lapatinib and sulforaphane synergistically led to a decline in cell viability, which was better than the effect of each agent alone. It is consistent with the efficiency of lapatinib combined with one of isothiocyanates (sulforaphane, erucin, or sulforaphene) in BC, suggesting sulforaphane sensitize lapatinib-resistant cells to the drug [25]. Meanwhile, Angelika observed that combination of lapatinib with sulforaphane caused a decline in cell invasion [25]. Our study confirmed it and showed lapatinibsulforaphane was effective in inhibiting invasion of SGC-7901 and lapatinib-resistant SGC-7901 cells.
In cancer cells, inhibition of cell cycle regulation is a potential cancer treatment. Previous studies indicated that sulforaphane suppressed the proliferation of cancer cells by inducing G1 or G2/M arrest in multiple cancer cells [26]. YUNG reported sulforaphane inhibited proliferation of AGS human GC cells by inducing the cellular portion of the G2/M phase, whereas lapatinib was only caused in a significant G1 arrest in lapatinib-sensitive BC cell [7]. In the present study, lapatinib significantly induced G0/G1 arrest in SGC-7901 cell and sulforaphane led to a decrease of S phase in SGC-7901 and lapatinib-resistant SGC-7901 cell. e combination of lapatinib and sulforaphane resulted in an accumulation of cells in the G0/G1 phase and a reduction in the S phase of both cells.
is is the first time to confirm that lapatinib-sulforaphane regulated cell cycle in SGC-7901 and lapatinibresistant SGC-7901 cells.
Apoptosis is a physiological process that regulates cell death. Inducing apoptosis is one of the common methods to control cancer progression. Lapatinib inhibited the growth of HER2 gene-amplified trastuzumab-sensitive and resistant GC cells by inducing apoptosis [27], whereas lapatinib had no impacts on lapatinib-resistant BC cells [7]. Meanwhile, sulforaphane was reported to induce apoptosis of cancer cells through AMPK-dependent or SHH pathway [28,29]. Our results were similar to previous researches. We found lapatinib induced apoptosis in SGC-7901 cell, and sulforaphane had significantly impact on both cells. e combination of lapatinib and sulforaphane was the most effective agent to inducing apoptosis of SGC-7901 and lapatinibresistant SGC-7901 cells. It indicates that sulforaphane could enhance the effects of lapatinib on apoptosis in drug-sensitive and drug resistant cells.
To investigate the functional mechanism of lapatinib combined with sulforaphane on GC cells, the expressions of HER2 signaling pathway related genes were measured. Akt and ERK were the main HER2 downstream effectors, which played an important role inPI3/AKT and MAPK signaling pathway to regulate the growth, apoptosis, and death of cells.
In this study, lapatinib significantly downregulated the expressions of p-HER2, p-AKT, and p-ERK, but lapatinib reduced the expressions of HER2, AKT, and ERK in lapatinib-resistant cells. It revealed that the drug resistance of SGC-7901 cell may be related to HER2, AKT, and ERK proteins expression. On the other hand, lapatinib-sulforaphane treatment significantly inhibited PI3/AKT and MAPK signaling pathway, especially in the decreased expressions of p-HER2, p-AKT, and p-ERK. e similar results were also shown in BC and HER2-positive GC [4,25].
In conclusion, we investigated the molecular mechanism of lapatinib combined with sulforaphane in SGC-7901 and lapatinib-resistant SGC-7901 cells. Our results suggested that lapatinib-sulforaphane might be a novel and promising agent in both lapatinib-sensitive and lapatinib-resistant GC patients.

Data Availability
All data are available within the article.