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Anticancer effect of the traditional Chinese medicine herb Maytenus compound via the EGFR/PI3K/AKT/GSK3β pathway

Background Cancer is a leading cause of death worldwide; folk anticancer medicinal plants have applied for cancer treatment. The Maytenus compound tablet as traditional Chinese compound medicine has been approved for alleviating hyperplasia of mammary glands, whether it can inhibit cancer still unknown. The study was to evaluate the anticancer activity of the Maytenus compound tablet. Methods MTS assay evaluated the anti-proliferation effect of the Maytenus compound on H226, A2058, 786O and HeLa cancer cells and two normal epithelial cell lines, namely, 16HBE and Hecate. Nude mouse xenograft tumor model using H226 and HeLa cells examined the drug’s anticancer effect in vivo. Western blot assay studied the possible mechanism. Results The Maytenus compound indicated obvious ability to against proliferation in four strains of cancer cells, particularly against H226 cells by an IC50 of 85.47±10.06 µg/mL and against HeLa cells by an IC50 of 128.74±17.46 µg/mL. However, it had a low cytotoxicity in human normal epithelial cell lines 16HBE with an IC50 of 4,555.86±25.21 µg/mL and Hecate with an IC50 of 833.56±181.88 µg/mL. The Maytenus compound at the 2.45 g/kg oral dosages inhibited the proliferation of H226 cells and HeLa cells in nude mouse with inhibitory rates of 36.06% and 26.45%, respectively, and no organ toxicity. The Maytenus compound could significantly downregulate the expression of pEGFR, pPI3K, pAKT, pGSK3β, β-catenin, and c-MYC and upregulate the protein expression of GSK3β. Conclusions The Maytenus compound has significant anticancer activities against human cancer H226 and HeLa cells both in vitro and in vivo, highlighting it may be an anticancer medicine.


Introduction
Cancer mortality rate ranks the first around world and has become the major public health problem (1). Although there are many ways to treat cancer, it is still a difficult global problem to overcome. Nowadays, traditional anticancer medicinal plants have drawn growing interest as novel drugs because more and more studies have found that they have potentiality been as medicines for cancer prevention and therapy (2).
The Maytenus is used in Chinese traditional medicine for therapy of digestive inflammatory disorders such as ulcers (3,4), and it has antiparasitic activities (5). In recent years, the leaf extract of Maytenus in Brazilian has been reported to possess anticancer activity (6). Previously, maytansine, the chemical constituent of Maytenus, played important roles in anticancer effect by affecting micro-tubules (7,8). T-DM1 (Trastuzumab emtansine) including DM1, one of the maytansine derivative, has strong effect against HER2positive breast cancer as a novel drug (9). Trichothecin (TCN), isolated from an endophytic fungus of Maytenus, can induce G0/G1 cell cycle arrest and activate proapoptotic proteins, promoting cancer cell apoptosis (10).
The Maytenus compound tablet has been approved for alleviating hyperplasia of mammary glands [approval number: Dian drug (Z)05J02424)] in China, and it is a traditional Chinese compound medicine, there are no reports about its anti-cancer function. Therefore, the goal of this study was to evaluate the anticancer activity of the Maytenus compound tablet in both vitro and in vivo, further investigate the underlying mechanism.

Cell proliferation
MTS assay was also used to quantify cell proliferation. In brief, 2,000 cells and 150 μL medium were seeded each well in 96-well culture plates, and each group had six duplicate wells, incubated in complete medium with different concentrations of the Maytenus compound. Over a 4-day period, every 24 h, each well was added 30 μL of MTS, and full wavelength scanner was used to measured absorbance of each sample at 490 nm after they were incubated in a cell incubator for 2 h.

Clonogenic assays
One thousand and five hundred cells and 1.5 mL medium were seeded each well in 6-well culture plates each group had three duplicate wells. After cells were seeded for 24 h, they were treated with the different concentrations of Maytenus compound for 72 h. Then, cells were incubated continuously without the drug effect for approximately 10 days. After PBS washed cells three times, methanol fixed cells for 20 min and hematoxylin stained cells for 5 min, then cells were washed clean with ddH 2 O, aired, and naked clones were counted. All the isolated tumor tissues were cut into 4μm sections by fixing in the formalin and embedding in paraffin, sections were baked at 70 ℃ for 1 h, immersed in xylene for 20 min, and rehydrated in alcohol, followed by staining with hematoxylin-eosin.

Western blotting
Cells treated with the Maytenus compound for 72 h were collected and lysed completely in cold RIPA buffer (89900, Pierce, USA), BCA protein assay kit (23227, Thermo Fisher, USA) was measured protein concentration.

Statistical analysis
Data are expressed as the mean ± SD (standard deviation) and evaluated by independent t-test with SPSS version 18.0 software and Prism 5 (GraphPad Software, USA). The differences were considered statistically significant at P<0.05.

In vitro antitumor activity
Proliferation inhibitory effect at 72 h against four different human cancer cell lines (H226, A2058, 786O, HeLa) and human normal epithelial cell lines (16HBE, Hecate) were detected by MTS assay. As shown in Figure 1A, the Maytenus compound inhibited the growth of the four cancer cell lines in a dosedependent manner. In addition, the Maytenus compound revealed a stronger proliferation inhibitory effect of H226 cells with an IC50 of 85.47±10.06 μg/mL and of HeLa with an IC50 of 128.74±17.46 μg/mL than of 786O cells with an IC50 of 173.55±5.34 μg/mL and of A2058 cells with an IC50 of 227.40±27.20 μg/mL, suggesting that the Maytenus compound could exhibit a favorable anti-lung cancer and anti-cervical cancer effect. However, the Maytenus compound had low cytotoxicity in the human normal epithelial cell lines 16HBE with an IC50 of 4,555.86±25.21 μg/mL and Hecate with an IC50 of 833.56±181.88 μg/mL ( Figure 1B). According to the IC50 values of the Maytenus compound in the four cancer cell lines at 72 h, we chose the H226 and HeLa cells lines to continue the study of antitumor activity of the Maytenus compound. Then, we selected 0, 30, 60 and 120 μg/mL Maytenus compound concentration to treat H226 cells and selected 0, 50, 100 and 200 μg/mL concentration to treat HeLa cells.

The Maytenus compound had anti-activity effects on H226 and HeLa cells
After H226 and HeLa cells were treated as mentioned above, MTS assay was detected the cell viabilities. As shown in Figure 2A, the Maytenus compound inhibited the cell growth significantly, the cell viabilities of H226 and HeLa cells were decreased with the increased in Maytenus compound concentration and were decreased with the increased in Maytenus compound treatment time. In addition, as shown in Figure 2B,

Clonogenic assays
As shown in Figure 3, after H226 and HeLa cells were treated with the Maytenus compound for 72 h, the cells' clone formation capacity was reduced significantly compared with that of the control cells. Additionally, the clone numbers of the H226 and HeLa cells decreased with the increase in the Maytenus compound concentration. It indicates that the Maytenus compound had anti-clonogenic cancer cell ability.

In vivo antitumor activity
Nude mice were intragastrically administered the Maytenus compound at doses of 2.45 g/kg every day. As shown in Figure 4, the Maytenus compound inhibited H226 cells and HeLa cells tumor growth in the mouse models. Compared to control group, the inhibition effects in the experiment group were significant (P<0.05), the inhibition rates for tumor growth were 36.06% and 26.45% for the H226 mouse models and HeLa mouse models, respectively ( Table 1). The tumor weight results also indicated the antitumor activity of the Maytenus compound in vivo. Furthermore, the Maytenus compound had no significant effect on the mouse bodyweight, liver index, spleen index and kidney index. These results clearly showed that the Maytenus compound had a significant anticancer effect in vivo.

HE staining analysis
As shown in Figure 5, in the control groups, the tumor cells volume was large and round, nucleoli large and distinct, the cell structure was clear and intact, suggesting good growth. However, in the Maytenus compound groups, the tumor cells showed degenerative changes, karyopyknosis and fibrosis; in the solid tumor from the HeLa Maytenus compound groups, necrosed area could be found, where the structure showed loss of nuclei and fragmentation of

Western blot analysis
To further understand the antitumor activity of Maytenus compound on H226 and HeLa cells, western blot was used to analyze proteins change in cells treated with the Maytenus compound for 72 h. As shown in Figure 6, the Maytenus compound reduced the level of EGFR, pEGFR, pPI3K, pAKT, pGSK3β, β-catenin and c-MYC protein and increased protein expression of GSK3β in a dose-dependent manner. These results indicated that the Maytenus compound could inhibit proliferation of H226 and HeLa cells by down-regulating the phosphorylation levels of EGFR, PI3K, AKT and GSK3β.

Discussion
Numerous valuable "treasures" from Chinese traditional medicine were discovered because of the development of science and technology, especially national herbal medicine (12,13). Some Chinese traditional medicine substances have been proven to possess anticancer properties without side effects, such as dihydroartemisinin (14). In addition, in China, Chinese herbal medicine products as antitumor agents have thousand-years of history. In our study, the antitumor activity of the Maytenus compound against four cancer cell lines, including H226, A2058, 786O and HeLa, were confirmed in a dose-dependent manner, and these effects on H226 and HeLa were also confirmed in a timedependent manner. Additionally, the Maytenus compound showed low toxicity in human normal epithelial cells, and the results are consistent with those of Nkosinathi, who reported that the methanolic root extracts of Maytenus procumbens exhibited moderate cytotoxic effects on the cancer and weak cytotoxicity in the normal cells (15). In addition, the Maytenus compound had the ability to control H226 and HeLa cell proliferation in nude mice in vivo without cytotoxicity. The result indicated that the Maytenus compound could be a potential natural and novel anticancer drug, especially for lung cancer and cervical cancer. Lung cancer has high morbidity and mortality worldwide (1). Cervical cancer is the fourth in cancer related mortality among all cancer types and second in cancer related mortality among females (16,17), highlighting the importance of studying the antitumor mechanism of the Maytenus compound. Some reports have shown that some extracts of Maytenus have an antitumor mechanism, such as pristimerin, a natural triterpenoid isolated from Celastrus and Maytenus spp, which has been found to inhibit tumor cells in vitro and in vivo, and affect the proliferation, survival, angiogenesis and metastasis though the NF-kB, MAPK/ERK and AKT/mTOR pathways (18). Additionally,  the methanol extract of Maytenus royleanus leaves and its fractions possess anti-proliferative activity against prostate cancer by exhibiting cleavage of Caspase-3 and PARP (19).
As the derivative of maytansine, T-DM1 combined with pertuzumab significantly inhibited the phosphorylation of HER3 or EGFR, and its downstream factor AKT inhibited gastric cancer proliferation (20). The EGFR is a tyrosine kinase receptor that belongs to the Erb family, which is overexpressed in many tumor types, including lung cancer and cervical cancer (21,22). EGFR plays an important role in the physiological processes of the development and maintenance of epithelial tissues, and upregulated EGFR expression can activate cell proliferation and differentiation signaling pathways that lead to the progression of cancer, increase the metastasis and the suppression of apoptosis and angiogenesis (23)(24)(25). Drugs that affect the activity of EGFR have been used in clinical practice for cancer therapy (26,27). The major molecular mechanism of EGFR-induced tumor progression is that EGFR kinase phosphorylation activates the PI3K/ AKT signaling pathway (28). PI3K phosphorylates the 3-hydroxyl of phosphatidylinositol, regulating proliferation, survival and metastasis signaling pathways, which are important for cancer (29)(30)(31). AKT (AK mouse plus transforming or thymoma), as a downstream component of the PI3K pathway, is a fundamental protein mediating signals from the receptor tyrosine kinases family, it is a frequent oncogene (32,33). Consistent with these data, western blot was used to evaluate the expression of the EGFR, pEGFR, PI3K, pPI3K, AKT, and pAKT proteins in the cells with the Maytenus compound treatment, and we obtained evidence that the Maytenus compound reduced the levels of the EGFR, pEGFR, PI3K, pPI3K, AKT, and pAKT proteins. These data collectively indicate that the Maytenus compound triggers the EGFR/PI3K/AKT signaling pathways.
The underlying downstream mechanism of the Maytenus compound in inhibiting the proliferation of cancer cells though the EGFR/PI3K/AKT signaling pathways still need to elucidate furtherly, many studies have reported that PI3K/AKT is the upstream regulatory pathway of the GSK3β/β-catenin protein combination related with cell proliferation (34)(35)(36). Activated AKT translocates to the cell membrane, phosphorylating serine/threonine kinase glycogen synthase kinase 3β (GSK3β) (37,38). Under normal physiological conditions, GSK3β is highly expressed, and its main role is to destroy the complex containing APC and Axin, promote the decomposition of β-catenin and weaken the stability of β-catenin; however, GSK3β is less expressed in cancer cells, which promotes the stability of β-catenin (39,40). Then, β-catenin can enter the nucleus and activate its target genes, including c-MYC (41). Additionally, we found that the Maytenus compound significantly inhibited the expression of pGSK3β, β-catenin and c-MYC and promoted the expression of GSK3β.
In conclusion, the Maytenus compound is a Chinese six-herb medicine, which possesses favorable inhibitory activity against the growth of human H226, A2058, 786O and HeLa cancer cells in vitro, especially against H226 and HeLa. The Maytenus compound inhibits the growth of H226 and HeLa xenografts in vivo.
In addition, we found that the Maytenus compound played a role in anticancer activity via the EGFR/PI3K/AKT/GSK3β pathway, and many of the traditional Chinese medicines have been shown to have anticancer activity through multiple targets (42,43); therefore, the Maytenus compound might have other pathways. Next, we further studied its anticancer mechanism. Maytansine has a high inhibition effect on cancer, but using it alone has hepatotoxicity, and it has been assessed in several oncology clinical trials (44). However, in this study, the Maytenus compound had a low cytotoxicity in human normal epithelium cells and no toxicity in mouse organs. Therefore, the Maytenus compound could be a potential treatment agent for lung cancer and cervical cancer. However, before it can proceed further in preclinical tests, considerations such as safety evaluations, pharmacokinetics and a suitable oral administration dosage should be investigated.

Conclusions
Taken together, our results indicate that the Maytenus compound has significant anticancer activities against human cancer H226 and HeLa cells both in vitro and in vivo, highlighting it could be a novel potential anticancer drug. org/10.21037/tcr.2019.09.30). The authors have no conflicts of interest to declare.
Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. This study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). The animal experiments were performed in accordance with the institutional ethical requirements of the Kunming Medical University and approved by the Ethics Committee of Kunming Medical University (No. KMMU2019060). Individual informed consent was waived.
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