Glucosylated Epigallocatechin Gallate (EGCG) Derivatives Combined with EGFR Tyrosine Kinase Inhibitor Overcome Resistance in EGFR T790M Mutant Lung Cancer

Glucosylated Epigallocatechin Gallate (EGCG) Derivatives Combined with EGFR Tyrosine Kinase Inhibitor Overcome Resistance in EGFR T790M Mutant Lung Cancer. Biomed abstract EGFR tyrosine kinase inhibitors (TKIs) gefitinib and erlotinib are approved treatments for non-small cell lung cancers harboring activating mutations. Unfortunately, treatment with tyrosine kinase inhibitors for a period of time will result in drug resistance, most frequently due to the secondary T790M mutation. Epigallocatechin gallate (EGCG) is the most abundant and bioactive catechin in green tea, it has a strong inhibitory effect on various cancers. We synthesized and identified two glucosylated EGCG derivatives. In this study, we found that the stability and transepithelial transport rate of EGCG derivatives was improved compared with EGCG. Glucosylated EGCG-G2 treatment at different concentrations significantly inhibited the cell growth of EGFR L858R/T790M mutant cells. It was found in vitro that EGCG-G2 combined with gefitinib significantly inhibited the proliferation. More importantly, EGCG-G2 combined with gefitinib downregulate EGFR phosphorylate in NCI-H1975 cells. Taken together, our study suggests that combination of EGFR-TKI and EGCG derivatives maybe a potential treatment strategy to conquer drug resistance for acquired resistant NSCLC. In this study, we further studies regarding the biological activity and bioavailability of EGCG glycosides. In the current study, we explored the antitumor effect of EGCG glycosides on NCI-H1975 which is an acquired EGFR-TKI resistant cell line with T790M and L858R mutations. We investigate whether EGCG glycosides combined treatment with EGFR TKIS could synergistically inhibit NCI-H1975 cell growth by through suppressing phosphorylation of EGFR. Mean-while, we established a caco-2 cell monolayer model and simulated the absorption of EGCG and EGCG glycosides in the intestinal tract. Our current studies suggest that the combination of EGCG glycosides and EGFR TKIs may provide a potential strategy for NSCLC.

bodies plays a key role in NSCLC treatment [4][5]. Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs), such as gefitinib and erlotinib have exhibited remarkable therapeutic effects against NSCLC with exon 19 deletions and L858R point activating mutations in EGFR, but resistance arises rapidly in all patients after varying periods of time [6][7]. Most frequently due to the secondary T790M mutation within the ATP-site of the receptor, which has been detected in 50% of NSCLC cases with acquired resistance [8].
Due to the limited treatment options available for individuals with advanced lung cancer, the novel strategies to conquer drug resistance is an emergency to prolong overall survival time of NSCLC patients.
Epigallocatechin gallate (EGCG) is the most abundant component and most active phenolic constituent of green tea catechins, has been extensively studied in various cancers such as liver cancer, lung cancer, breast cancer, throat cancer, prostate cancer and bladder cancer [9][10]. EGCG also inhibited EGFR tyrosine kinase activity in A431 human epidermal carcinoma cells [11]. EGCG inhibits the phosphorylation of EGFR and is considered to be an important anti-tumor mechanism of EGCG. In our previous study, two novel EGCG glycosides (EGCG-G1 and EGCG-G2) were chemo selectively synthesized by a chemical modification strategy. The EGCG glycosides are more stable than EGCG in aqueous solutions and the EGCG glycosides exhibited increased water solubility: EGCG-G2 and EG-CG-G1 were 15 and 31 times as soluble EGCG, respectively [12]. In this study, we further studies regarding the biological activity and bioavailability of EGCG glycosides. In the current study, we explored the antitumor effect of EGCG glycosides on NCI-H1975 which is an acquired EGFR-TKI resistant cell line with T790M and L858R mutations. We investigate whether EGCG glycosides combined treatment with EGFR TKIS could synergistically inhibit NCI-H1975 cell growth by through suppressing phosphorylation of EGFR. Meanwhile, we established a caco-2 cell monolayer model and simulated the absorption of EGCG and EGCG glycosides in the intestinal tract.
Our current studies suggest that the combination of EGCG glycosides and EGFR TKIs may provide a potential strategy for NSCLC.

Cell Cultures
The human NSCLC cell lines NCI-H1975 and the human colorectal adenocarcinoma cell line Caco-2, were obtained from the Amer-

MTT Assay
Cell viability of NSCLC cell line was evaluated by MTT assay.
NCI-H1975 cells were seeded in 96-well plates at a density of 2 × 104 cells/well overnight and then treated with EGCG derivatives (0, 10, 20, 40, 60, 80 or 150 µM), EGFR TKIs alone (1µM), or a combination treatment of EGFR TKIs (1μM) and EGCG derivatives (30µM) for 48 hours. Then added 20µL MTT (5mg/mL in phosphate buffered saline) to the each well, and the plates were incubated for 4 h at 37˚C. The culture medium was aspirated and 150μLdimethyl sulfoxide was added to dissolve the formazan crystals. The optical density (OD) was measured at 492 nm using a microplate reader.
The percentage of inhibition was calculated as follows:

Western Blot Analysis
After treatment, NCI-H1975 cells were lysed in RIPA buffer containing PMSF (protease and phosphatase inhibitors) and

Preparation of Caco-2 Monolayers
Caco-2 cells were grown as epithelial monolayers by seed-

EGCG And EGCG Derivatives Transport Assay
Before initiating the transport experiments, the monolayers were washed twice with warm (37°C) HBSS. The incubation buffer on both sides of the monolayers was then removed by aspiration.
EGCG and EGCG-G2 in 0.5 mL HBSS was added to the AP side or

The Apparent Permeability (Papp)
The apparent permeability coefficients (Papp) was expressed in cm/second and was calculated by, P"app"="dQ" /"dt" ×1/(A⋅C0) Where dQ/dt is the amount of solutes transported across the Caco-2 barrier in time dt, C 0 is the initial drug concentrate, and A is the cross-sectional area of the epithelium in contact with apical solution.

Results and Discussion
The Synthesis of EGCG-G1 (2)

Signaling
The EGFR signal pathway is a crucial target in NSCLC treatment [7]. To characterize whether the growth inhibition induced by EG-CG-G2 and gefitinib might involve EGFR signaling, we examined the phosphorylation levels of the members of the Her (ErbB) family of receptor tyrosine kinases, which includes EGFR (ErbB1), Her2 (ErbB2/Neu), Her3 (ErbB3), and Her4 (ErbB4). NCI-H1975 cells were treated with EGCG-G2 and gefitinib alone or combination of two drugs for 12h. NCI-H1975 cells were stimulated with 10 ng/mL EGF for 10 minutes. As shown in Figure 4, in combination with gefitinib, EGGC-G2 inhibited the phosphorylation of EGFR family more significantly than the alone.

Figure 4:
Combination of EGCG-G2 and grlotinib has synergized inhibitory effects on the phosphorylation the four members of the Her (ErbB) family in H1975 cells. NCI-H1975 cells were treated with EGCG-G2 (30μM), gefitinib (1μM) or EGCG-G2 plus gefitinib, after 12 hours, NCI-H1975 cells were stimulated with or withnot 10 ng/mL EGF for 5 minutes. The expression levels of the proteins were determined by western blotting with β-tubulin as the loading control. Figure 5: Transport of EGCG and EGCG-G2 across Caco-2 monolayers. TEER values were also measured to account for the integrity of the monolayer during the experiment. A. TEER values of Caco-2 monolayer (Ω·cm 2 ). B. The transepithelial transport rate of EGCG and EGCG-G2 (50μg/mL) in the apical to basolateral direction. C. The transepithelial transport rate of EGCG and EGCG-G2 (50μg/mL) in the basolateral to apical direction. *** p < 0.001.

Transport of EGCG and EGCG-G2 Across Caco-2 Cell Monolayers
According to the TEER value it was concluded that the integrity and tightness of epithelial cell monolayers were maintained after 21 days culturing [16]. To ensure monolayer integrity during the assay, all wells were monitored TEER values before and after the experiments. The TEER values did not exhibit a drop during the experiment, indicating that the cell monolayer was intact and the transport of EGCG and EGCG-G2 did not damage the monolayer ( Figure 5A). The transported amounts of EGCG and EGCG-G2 were assessed for both the apical to basolateral (AB) and basolateral to apical (BA) directions. As shown in Figure 5B and 5C, the amounts of EGCG-G2 transported in the acceptor chamber were higher in the BA direction than in the AB, and the uptake of EGCG-G2 in the BA direction was 5.7-fold higher than the corresponding values of EGCG at 60min. The apparent permeability coefficients (Papp) values of EGCG-G2 in the BA direction were 7.55 × 10−7cm/s and 3.2 × 10−7 cm/s were significantly higher than the AB direction.

Conclusion
In conclusion, glucosylated epigallocatechin gallate (EGCG) derivatives have improved stability and absorption compared to EGCG. EGCG-G2 has shown significantly inhibitory effect on EGFR L858R/T790M mutant NSCLC cells. In addition, EGCG-G2 combinated with gefitinib markedly inhibited EGF-induced EGFR phosphorylation Table 1. EGCG-G2, either alone or in combination with gefitinib has the potential in EGFR-TKI-resistant NSCLC treatment. Data are reported as mean ± SEM (n = 3).