Combination of Huanglian Jiedu Decoction and erlotinib delays growth and improves sensitivity of EGFR-mutated NSCLC cells in vitro and in vivo via STAT3/Bcl-2 signaling

Abstract

Erlotinib, an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI), is widely used in the treatment of non-small cell lung cancer (NSCLC). However, erlotinib resistance leads to high mortality in patients with NSCLC, while the activation of STAT3 is closely related to erlotinib resistance. Studies have shown that the main components of Huanglian Jiedu Decoction (HJD) have antitumor effects. Therefore, the anticancer effect of HJD combined with erlotinib on NSCLC cells was investigated. The NSCLC HCC827, HCC827ER, and H1975 cell lines as well as xenograft nude mice were selected as models to study the effects of HJD. The proapoptotic effects of HJD were examined by CCK-8 and apoptosis assays. ELISA, immunostaining, and western blot analysis were also performed. HJD considerably enhanced the anticancer effect of erlotinib in both EGFR-TKI-resistant and -sensitive NSCLC cells. HJD promoted erlotinib-induced apoptosis and caspase 3 activity. The co-treatment also inhibited the expression of Bcl-XL, Bcl-2, and p-STAT3. In addition, siSTAT3 had similar functions with HJD. In particular, the apoptotic rates of erlotinib-stimulated HCC827, HCC827ER, and H1975 cells were enhanced by transfecting siSTAT3. Furthermore, overexpression of STAT3 significantly inhibited HJD-mediated erlotinib sensitization. The combined use of HJD with erlotinib significantly reduced tumor growth in erlotinib-resistant HCC827ER and H1975 ×enografts, induced caspase 3, and inhibited Ki67, STAT3, and Bcl-2 expression. HJD significantly alleviated erlotinib resistance by regulating the STAT3/Bcl-2 signaling pathway, which is a promising method to overcome the EGFR-TKI resistance of NSCLC.

Introduction

Lung cancer is one of the most frequently diagnosed malignant tumors and almost 80–85% of lung cancer is non-small cell lung cancer (NSCLC). Primary lung cancer is the leading cause of cancer-related death worldwide (1,2). Lung cancer is complemented with a high rate of mortality and is often not diagnosed until reaching a late stage. With advances in many aspects of the treatment, diagnosis, and classification, the overall survival of NSCLC patients at early-stage NSCLC has improved after complete resection. However, postoperative recurrence and metastasis are also major obstacles to prolonged survival in early-stage NSCLC (3–5). Even though conventional chemotherapy has favorable therapeutic effects, the side effects of the drugs that influence the life quality of patients greatly limit the application. As drug resistance is commonly seen with NSCLC cases, more effective treatment strategies should be explored.

The epidermal growth factor receptor (EGFR), a member of the transmembrane receptor tyrosine kinases of the ErbB family, is involved in the regulation of signal pathways, including proliferation, metastasis, epithelial-mesenchymal transformation, and apoptosis. Gefitinib (Iressa) and erlotinib (Tarceva) are two US FDA-approved EGFR-specific tyrosine kinase inhibitors (TKIs) that have exhibited clinical benefit in treating NSCLC (6,7). Erlotinib treatment prolonged the progression-free survival of NSCLC patients, who harbor EGFR exon 19 deletion or L858R mutations (EGFR Mut+ NSCLC) (8). However, almost all tumors eventually develop acquired resistance to the treatment, with 50% of the patients developing EGFR T790M resistance mutations (9). Several mechanisms of EGFR-TKI resistance have been reported, including EGFR T790M mutation and MET amplification. Overactivation of several protein kinases, such as signal transducer and activator of transcription 3 (STAT3), has been reported to be one of the causes of drug resistance in NSCLC (10). HCC827 cells carry the erlotinib-sensitive mutation (E746-A750 deletion), but not the drug-resistant mutation (T790M) (11). Subsequently, HCC827 cells are sensitive to erlotinib and other EGFR-TKIs (12). Interestingly, compared with HCC827 cells, erlotinib-resistant HCC827 cells (HCC827ER) showed higher levels of pSTAT3 (13). Erlotinib also induces resistance in PC-9 cells by activating STAT3 (14). In addition, HCC827ER cells showed greater invasiveness in Matrigel-coated Boyden chambers than the parental HCC827 cell line (15). H1975 is a human adenocarcinoma cell line with the EGFR T790M/L858R mutation that is regarded as an EGFR-TKI acquired drug-resistant cell line (16).

Huanglian Jiedu Decoction (HJD) is one of the traditional Chinese medicines used to treat diabetes in ancient China. It is known for its liver protective effects, including liver detoxification (17). It was first recorded in Wai-tai-mi-yao (Arcane Essentials from the Imperial Library) written by Wang Tao of the Tang dynasty and widely used in clinical practice (18). The main active components of HLD are known to have antitumor effects. For example, baicalin and baicalein can reduce STAT3 activity, further downregulate the expression of IFN-γ-induced PD-L1, and then restore the sensitivity of T cells to kill tumor cells (19). Berberine hydrochloride can inhibit the proliferation of cervical cancer cells HeLa229 and induce apoptosis by upregulating p53 and downregulating the expression levels of Bcl-2 and COX-2 mRNA (20). Baicalin inhibits cell growth of SW1353 by downregulating Bcl-2 and activating caspase-3 and −9 (21). In addition, wogonin (5,7-dihydroxy-8-methoxyflavone) (an O-methylated flavone) inhibited tumor proliferation in Raji xenograft mice by decreasing the expression of Ki67 (22). Therefore, we hypothesized that the STAT3/Bcl-2 signaling pathway and its downstream targets may be involved in the antitumor effect of HJD.

As erlotinib and HJD act via two different pathways key to tumor growth, using these drugs concurrently may confer promising clinical benefits to NSCLC patients. The aim of this study was to discuss the potential of combined inhibition in clinically relevant xenograft models of EGFR TKI resistance and to explore whether dual blockade may provide a significant advantage over monotherapy.

Materials and methods

Preparation and HPLC analysis of HJD

The herbal materials of HJD composed of Fructus gardeniae (90 g), Cortex phellodendri (60 g), Radix scutellariae (60 g), and Rhizoma coptidis (90 g). The herbs were all purchased from Ma'anshan Jingquan Traditional Chinese Medicine Decoction pieces, Co., Ltd. The authenticity of the plant species was confirmed by Dr Huang Yawei from Ma'anshan Jingquan Traditional Chinese Medicine Decoction pieces Co., Ltd. HJD was refluxed with water (1:10, w/v) for 2 h. Afterwards, the HJD extract was obtained by drying the concentrate with vacuum concentration. Powered HJD samples were sonicated with methanol for 1 h. The sample was finally filtrated through a 0.45 µM membrane filter prior to HPLC analysis. The separation was performed on a Waters Acquity C18 column (250×4.6 mm, 5 µm). Separation was carried out by acetonitrile (A) and 0.05% K₂HPO₄ and 0.05% triethanolamine in water (B). The linear gradient elution was 82% A, in 0–9 min; 82–73% A, in 9–27 min; 73–60% A, in 27–45 min; 60–82% A, in 45–60 min with a flow rate of 1.0 ml/min. The UV detection was operated at 254 nm, and the injection volume was 20 µl. The external standard method was applied for the identification by comparing their retention time and spectrum against known standards (Fig. 1 and Table I).

Figure 1.

HPLC chromatograms of (A) HJD extracts and (B) standards. Peaks: 1, geniposide; 2, baicalin; 3, berberine hydrochloride; 4, wogonoside; 5, baicalein; 6, wogonin.

Table I.

The structure of the standards in HPLC chromatograms of HJD extracts.

Name	Type	Structures
Geniposide	Iridoids
Baicalin	Flavonoids
Berberine hydrochloride	Isoquinoline alkaloids
Wogonoside	Flavonoids
Baicalein	Flavonoids
Wogonin	Flavonoids

Cell culture and reagents

The NSCLC HCC827, A549, H460, and H1975 cell lines were obtained from the American Type Culture Collection (Manassas). Cell lines were maintained in DMEM medium supplemented with 10% fetal bovine serum (FBS) at 37°C with 5% CO₂. The acquired erlotinib-resistant HCC827 cell line (HCC827/ER) was established by exposing erlotinib-sensitive HCC827 cells to gradually increasing concentrations of erlotinib from 0.1 to 10 µM over a 6-month period at 37°C. Erlotinib was purchased from Selleck (cat. no. S7786). The caspase-3 inhibitor Z-DEVD-FMK was obtained from Gene Operation (cat. no. IAP1201). Stock solution was prepared in dimethyl sulfoxide at 10 mM and was stored at −20°C.

Cytotoxicity assay

The Cell Counting Kit-8 (CCK-8) assay was used to determine cell proliferation. Cells (5×10⁴) were prepared and mixed evenly, and then 0.1 ml cell suspension was added to each of the 96-well plates. Cells were cultured in a 5% CO₂ incubator at 37°C for 72 h. Subsequently, 10 µl CCK-8 solution was added to each well and incubated for 4 h (in the dark). Cell proliferation was evaluated by using FLUOstar^® Omega microplate reader to measure the absorbance of cells at the wavelength of 450 nm.

Detection of cell apoptosis by flow cytometry

Apoptosis was detected using the apoptosis detection kit (Biosea Biotechnology). The HCC827, H1975 and HCC827ER cells were separately inoculated on a 6-well plate and cultured in complete medium for 72 h, then treated with DMSO (control), HJD only, erlotinib only, and HJD+E, respectively. The cells were collected, washed twice in cold PBS, then stained with Annexin V and propyl iodide (PI). The stained cells were collected and analyzed by flow cytometry. FlowJo software was used to analyze the results.

Caspase-3/7 activity assay

Caspase-3/7 activity was measured by the Apo-One homogeneous caspase-3/7 Assay kit (Promega, no. G7790). Then, 1×10⁴ cells per well were inoculated, incubated for 24 h and then treated with HJD, erlotinib and HJD+E, respectively, for 72 h. The cells were then washed with PBS three times. The activity of caspase-3/7 was evaluated according to the manufacturer's instructions.

Cell lysates preparation and immunoblotting

The cells were mixed with RIPA lysis buffer to extract total protein. The protein concentration was measured by bicinchoninic acid (BCA) method. Then protein samples (50 µg) were subjected to 10% SDS-PAGE gel electrophoresis and transferred to PVDF membranes. Subsequently, PVDF membranes were blocked in 5% skimmed milk for 1 h at room temperature and then incubated with primary antibody overnight at 4°C. Cleaved-caspase 3 (cat. no. ab49822, 1:1,000), p-STAT3 (cat. no. ab76315, 1:3,000), t-STAT3 (cat. no. ab68153, 1:2,000), Bcl-2 (cat. no. ab182858, 1:2,000), Bcl-XL (cat. no. ab32370, 1:1,000), GAPDH (cat. no. ab8245, 1:5,000), p-STAT1 (cat. no. ab109461, 1:3,000), t-STAT1 (cat. no. ab180814, 1:1,000), p-STAT2 (cat. no. ab53132, 1:3,000), t-STAT2 (cat. no. ab32367, 1:2,000), p-STAT5 (cat. no. ab32364, 1:2,000), t-STAT5 (cat. no. ab32364, 1:1,000) were purchased from Abcam. Next day, the membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG H&L (cat. no. ab205718; 1:6,000, Abcam) or goat anti-rat IgG H&L (cat. no. ab97057; 1:5,000, Abcam) secondary antibodies at room temperature for 2 h. Finally, the protein bands were detected using the Tanon-5200 Multi chemiluminescent gel imaging system.

In vivo study protocol

Six-week-old male nude mice were purchased from the Model Animal Reasearch Center of Nanjing University (Nanjing, China). Twenty-four mice (22 g) were maintained in a specific pathogen-free environment with a 12-h light/dark cycle and fed with standard food and water ad libitum. This study and the animal experimental protocols were approved by the Institutional Animal Care and Use Committee of Jiangsu Provincial Academy of Chinese Medicine.

Approximately 5×10⁶ HCC827/ER cells or H1975 cells were suspended in 0.2 ml DMEM medium and injected subcutaneously into the right flank region of nude mice, respectively. When the average tumor size reached ~75 mm³, the mice were randomized into four groups (n=6 each): i) Vehicle group, ii) HJD group (50 mg/kg), iii) erlotinib group (20 mg/kg), iv) combination of HJD (50 mg/kg) and erlotinib (20 mg/kg). All of the treatments were orally administrated once a day for 27 consecutive days. The formula, 1/2(length × width²), was used to estimate tumor sizes. The body weight and the tumor volume (V) were recorded every 3 days. At the end of the experiment, the mice were sacrificed by manual cervical dislocation, which resulted in euthanasia within approximately 10 sec. The tumors were then dissected and weighed to calculate the inhibitory rate.

The animal experiments were approved by the ethics committee of the Affiliated Hospital of Integrated Traditional Chinese and Western Medicine (Nanjing, China).

Immunohistochemistry

Tissues were collected and fixed in 10% formalin for 24 h at room temperature, then embedded in paraffin, and sectioned. The tissue sections (5 µm) were dewaxed, incubated with 3% methanol-hydrogen peroxide and washed with PBS three times. Sections were placed in citric acid buffer, heated and boiled, and washed with PBS. Then, the sections were blocked with goat serum (cat. no. 7481; Abcam) for 20 min, and incubated with STAT3 and Bcl-2 antibodies at 4°C overnight. Next day, the sections were incubated with goat anti-rabbit IgG H&L (horseradish peroxidase) antibodies (cat. no. ab205718; 1:5,000, Abcam). A 3,3′-diaminobenzidine (DAB) kit was used to observe the specific labeling (brownish yellow staining), and hemotaxylin was used for counterstain cell nuclei (blue staining). Images were obtained from each slide using a digital trinocular microscope (Zeiss Axio Observer A1).

H&E staining

The collected nude mouse tissues were fixed with formalin, paraffin-embedded, cut into 4 µm slices, and roasted in an oven for 3 h. Sections with xylene were first washed three times, then different gradient ethanol (75, 85, 95 and 100%) was used to rinse the tissues twice, prior to staining the sections with hematoxylin. Then, sections were immersed in hydrochloric acid-ethanol solution, dyed with ammonia water, and redyed with eosin. Finally, sections were immersed in different gradient of ethanol (75, 85, 95 and 100%) and sealed with neutral resin.

Statistical analysis

SPSS Statistics version 13.0 was used for all statistical analyses (SPSS Inc.). Statistical comparisons were performed using one-way ANOVA followed by Tukey's multiple comparison post hoc test and P<0.05 was considered statistically different. The results were presented as the mean ± SD.

Results

HJD strengthens EGFR-TKI sensitivity and reduces the drug resistance of NSCLC cells to erlotinib

To evaluate whether HJD could overcome the resistance of NSCLC cells to erlotinib, the inhibitory effect of co-treatments on HCC827, HCC827ER, H1975, H460 and A549 cell growth was assessed using a standard CCK-8 assay. The mutation profile of NSCLC cell lines is shown in Table II. Consistent with earlier studies, cells bearing mutated EGFR (HCC827) were more sensitive to erlotinib than those bearing wild-type EGFR (H460, A549). However, H1975 cells with both L858R and T790M mutations were relatively insensitive to erlotinib treatment. HCC827 erlotinib-resistant cells (HCC827ER) were established by a series of stepwise increase of the erlotinib concentration until HCC827ER was no longer responsive to 5 µM erlotinib. HCC827ER cells were more resistant to erlotinib than HCC827 cells (IC₅₀=31.26 vs. 0.11 µM) (Fig. 2A and B).

Table II.

Mutation profile of NSCLC cell lines.

Cell lines	EGFR gene	K-Ras gene
A549	WT	G12S
H1975	Exon19 T790M-L858R	WT
HCC827	Exon19 (E746-A750) del	WT
HCC827ER	Exon19 (E746-A750) del	WT
H460	WT	G61H

Figure 2.

The inhibitory effect of HJD and/or erlotinib on NSCLC cell lines after 72 h treatment with CCK-8 assays. (A) HCC827 cell viability after erlotinib treatment. (B) Different susceptibility of NSCLC cell lines (A549, H460, H1975 and HCC827ER) to erlotinib after 72 h treatment. *P<0.05 (vs. control). (C) Viability of NSCLC cell lines after 72 h treatment of HJD. (D-F) The inhibitory effect of HJD plus erlotinib on HCC827, H1975, and HCC827ER cells after 72 h treatment. *P<0.05 (vs. erlotinib); **P<0.01 (vs. erlotinib). Each value represents the mean ± SD.

To determine whether HJD can inhibit the growth of NSCLC cells, cell viability was determined. HJD (0–100 µg/ml) did not produce significant toxicity in NSCLC cells, independent of the EGFR-mutated status (Fig. 2C). However, HJD acting on NSCLC cells showed synergistic effects with erlotinib. The combination of HJD (50 µg/ml) with erlotinib led to 57% inhibition on the ability of HCC827ER cells (Fig. 2D), although erlotinib (10 µM) alone exhibited a slight effect (~20% inhibition) on cell ability (Fig. 2E). In parallel, the H1975 cell line was also sensitive to the combined administration (Fig. 2F).

HJD amplifies erlotinib-induced apoptosis

Apoptosis is involved in the anticancer effect of erlotinib (23). To measure whether the synergistic effect of HJD on erlotinib was associated with apoptosis, cells were stained with Annexin V-FITC/PI and analyzed using flow cytometry. As shown in Fig. 3A, erlotinib-stimulated apoptosis was significantly enhanced by HJD in H1975, HCC827 and HCC827ER cells (Fig. 3A). As demonstrated in ELISA assays, Caspase-3/7 activation was also significantly induced by combination treatment in these cell lines (P<0.05, Fig. 3B), suggesting that erlotinib-augmented activation of caspase was meaningfully promoted by HJD. Moreover, the caspase-3 inhibitor Z-DEVD-FMK could almost abrogate the activation of caspase 3 caused by HJD (Fig. 3C).

Figure 3.

HJD and erlotinib induced apoptosis in NSCLC cells. (A) Representative image for cell apoptosis stained with Annexin V-FITC/PI. HCC827, HCC827ER and H1975 cells processed with HJD, erlotinib, or HJD+E. The cells were then stained with Annexin V-FITC/PI and analyzed by flow cytometry. *P<0.05, **P<0.01 (vs. erlotinib). Each value represents the mean ± SD. (B) The effects of HJD, E and HJD+E on caspase-3/7 activity were determined by an ELISA kit. *P<0.05 (vs. erlotinib). (C) Each value represents the mean ± SD. The effects of HJD, E and HJD+E on caspase-3 activity evaluated by western blotting.

Erlotinib-induced STAT3 activation was inhibited by HJD

To better understand the drug resistance mechanism of HCC827ER cells, we carefully analyzed its characteristics. We found that the level of p-STAT3 in erlotinib-sensitive primary HCC827 cells was much lower than that in HCC827ER cells (Fig. 4A). Abnormal activation of STAT3 in tumor cells ensures the survival of malignant cells under stress conditions and also leads to multi-drug resistance (24). Erlotinib did not reduce the higher levels of STAT3 phosphorylation in HCC827ER cells (Fig. 4B). Therefore, we detected the effect of STAT3 on HJD-mediated erlotinib sensitization. Erlotinib therapy resulted in the feedback activation of STAT3 in H1975 cells (Fig. 4C), consistent with previous studies (13,25). However, HJD significantly inhibited the levels of p-STAT3 and its downstream targets, Bcl-2 and Bcl-XL proteins induced by erlotinib, suggesting that STAT3 may be related to HJD-mediated erlotinib sensitizing. In addition, we evaluated other members of the STAT family. The effect of Erlotinib and HJD was independent of the phosphorylation of STAT1, STAT2, and STAT5 (Fig. 4D). Furthermore, HJD did not potentiate the inhibitory effect of erlotinib on EGFR phosphorylation of HCC827ER and H1975 cells (Fig. S1).

Figure 4.

The effects of HJD, erlotinib, and HJD+E on the phosphorylation level of STAT3 and its downstream targets in HCC827, HCC827ER, and H1975 cells were detected by western blotting. (A) The expression of pSTAT3 and STAT3 in HCC728 and HCC827ER cells. (B and C) The expression of pSTAT3 and its downstream targets in HCC827ER and H1975 cells subjected to HJD/erlotinib alone or HJD plus erlotinib. (D) The protein levels of STATs in HCC827ER cells subjected to HJD/erlotinib alone or HJD plus erlotinib.

To verify the effect of STAT3 on erlotinib resistance, we transfected HCC827, HCC827ER, and H1975 cells with STAT3-specific siRNAs. The effect of siSTAT3 on apoptosis was assessed. The apoptosis rate of HCC827ER cells induced by erlotinib alone was 24.1±4.4%, and the apoptosis rate increased to 49.1±5.2% after siSTAT3 transfection (P<0.01). A similar increment in erlotinib-induced apoptosis was also observed in both H1975 and HCC827 cells after siSTAT3 transfection (Fig. 5A). siSTAT3 significantly inhibited endogenous STAT3 and enhanced the inhibition of erlotinib on H1975 and HCC827ER cell viability (Fig. 5B and C). Furthermore, plasmids expressing constitutively activated STAT3 (STAT3-C) were employed. Overexpression of STAT3 in H1975 cells significantly inhibited HJD-mediated erlotinib sensitization (Fig. 5D).

Figure 5.

The effect of siSTAT3 on apoptosis induced by HJD and erlotinib combination. (A) Flow cytometry was used to detect the effect of siSTAT3 on erlotinib-induced apoptosis of HCC827, HCC827ER and H1975. **P<0.01 (vs. erlotinib). (B and C) The effect of siSTAT3 on the viability of HCC827ER and H1975 exposed to erlotinib by CCK-8 assays. **P<0.01 (vs. erlotinib). (D) The effect of STAT3 plasmid on the viability of H1975 exposed to erlotinib and/or HJD by CCK-8 assays. **P<0.01 (vs. control). Each value represents the mean ± SD.

HJD potentiates the tumor-inhibiting activity of erlotinib in vivo

Next, we evaluated whether combination therapy could alleviate erlotinib resistance in HCC827ER and H1975 ×enograft nude mice. The nude mice were divided into the control, HJD, erlotinib, and HJD+erlotinib groups. After erlotinib treatment of HCC827ER xenograft, tumor growth was retarded. Although the tumor volume treated with HJD was smaller than that of the control group, there were no substantial differences. The combination of HJD with erlotinib in particular elicited an extremely strong tumor regression (Fig. 6). At the end of the experiment (27 days of co-treatment), tumors were barely palpable. In summary, these results indicate that erlotinib and HJD contributed to substantial tumor shrinkage of established xenografts bearing EGFR T790M mutation.

Figure 6.

Combined treatment of HJD and erlotinib inhibit tumor growth in mice-bearing HCC827ER or H1975 ×enografts. The nude mice (n=6) were given saline (control group), 20 mg/kg erlotinib, 50 mg/kg HJD or both, respectively. (A) Mice with representative HCC827ER tumors are shown. (B) Tumor volumes in mice-bearing HCC827ER xenografts. (C) Mice with representative H1975 tumors are shown. (D) Tumor volumes in mice-bearing H1975 ×enografts. Tumor volume was recorded every 3 days. Each value represents the mean ± SD.

Caspase 3-positive cells were greatly increased in the co-treatment group, whereas Ki67 expression was decreased in the co-treatment group, as shown in immunohistochemical assays (Figs. 7 and 8). The levels of STAT3 and its downstream signaling target, Bcl-2, were upregulated in the erlotinib group in mice-bearing HCC827ER tumors, compared to the saline-treated group (Fig. 7). As expected, the combination of HJD and erlotinib blunted the expression of STAT3 and Bcl-2 in mice-bearing H1975 ×enografts (Fig. 8). These results were also confirmed by western blot assays. For safety evaluation of the combinatorial treatment, the kidneys, liver, lungs and heart of nude mice were histologically analyzed. No obvious toxicity was observed in the main organs of mice-bearing xenografts subjected to HJD plus erlotinib treatment, as shown in Figs. S2 and S3. In vivo studies suggested that HJD could overcome TKI resistance by inhibiting the STAT3 signaling pathway. The STAT3/Bcl-2 pathway is involved in the in vivo antitumor effect of HJD combined with erlotinib.

Figure 7.

HJD inhibited erlotinib-induced STAT3/Bcl-2 activation in mice-bearing HCC827ER xenografts. Nude mice (n=6) were given saline (control group), 20 mg/kg erlotinib, 50 mg/kg HJD or both, respectively. (A and B) Immunohistochemistry was used to analyze protein levels of caspase 3, Ki67, and STAT3 in tumors of HCC827ER xenograft mice. The number of positive staining cells was estimated by a digital trinocular microscope. Each value represents the mean ± SD. (C) Western blotting was used to analyze protein levels of STAT3 and Bcl-2 in tumors of HCC827ER xenograft mice. *P<0.05 (vs. control).

Figure 8.

HJD inhibited erlotinib-induced STAT3/Bcl-2 activation in mice-bearing H1975 ×enografts. Nude mice (n=6) were given saline (control group), 20 mg/kg erlotinib, 50 mg/kg HJD or both, respectively. (A and B) Immunohistochemistry was used to analyze protein levels of caspase 3, Ki67, and STAT3 in tumors of H1975 ×enograft mice. The number of positive staining cells was estimated by an image analysis software. Each value represents the mean ± SD. (C) Western blotting was used to analyze protein levels of STAT3 and Bcl-2 in tumors of H1975 ×enograft mice. *P<0.05 (vs. control).

Discussion

Four members of the ERBB receptor family, namely EGFR, HER2, HER3, and HER4 are the most studied therapeutic targets for human malignancies (26,27). ErbB receptors induce EGFR activation by binding to ligands of different affinities (28). There are six major ligands for EGFR, namely epidermal growth factor, heparin-binding EGF, transforming growth factor, heregulin, amphiregulin, and betacellulin (29). In EGFR, when ligands bind to receptors, the receptors undergo conformational changes that produce homologous dimers and heterodimers (30), thereby activating downstream signaling pathways and promoting the development of cancer features, such as proliferation and survival, metastasis, and therapeutic resistance (28,31). Therefore, EGFR has become a new therapeutic target for cancer. EGFR inhibitors can be divided into two categories according to their sites of action: One is EGFR-TKIs, including erlotinib, afatinib, gefitinib and osimertinib (32,33). The other is extracellular EGRF-mab, such as cetuximab and panitumumab (34,35).

EGFR inhibitors have shown clinical benefit for NSCLC, but less than 10% of previously treated NSCLC patients have an objective tumor response (36,37) and patients develop resistance in the end. Erlotinib is an oral low-molecular-weight quinazoline-based agent, which acts as a reversible and selective kinase inhibitor of EGFR and has a better anticancer effect, less toxicity, and longer progression-free survival than chemotherapy (38,39). It has been applied for NSCLC patients with activating EGFR mutations. Although erlotinib is widely used in the treatment of NSCLC, many patients also develop resistance to it, which poses a challenge in oncology. Mutation of T790M in EGFR kinase is reported to be the most common mechanism of resistance to erlotinib (40). Our experiments show that T790M mutant H1975 cells are not sensitive to erlotinib, which is consistent with previous findings (41). The combination of HJD and erlotinib could overcome drug resistance induced by the T790M mutation in primary and acquired resistance cells in vitro and in vivo, which may represent an alternative therapeutic strategy in patients with acquired resistance to EGFR-TKIs.

The majority of components of HJD are known to influence critical processes of angiogenesis and metastasis by affecting numerous effector molecules. Findings of previous studies have shown that the main component of HJD has antitumor effects (42,43). The inhibitory effect of HJD on cancer cells has been confirmed in human myeloma cells, and its active compounds, baicalin and wogonin, are related to the inhibition of cancer cell growth, cell cycle arrest, and apoptosis (44,45). Results of this study revealed that HJD efficaciously increased the sensitivity of NSCLC cells to erlotinib. A synergistic effect was found in H1975 and HCC827ER cells. The combination displayed an additive effect on A549 cells bearing wild-type EGFR. In accordance with previous research (46,47), both HCC827ER and H1975 cells harbor the T790M mutation and exhibit a higher level of EGFR activity than A549 cells expressing wild-type EGFR (data not shown). It is widely acknowledged that T790M mutation is a critical factor for erlotinib resistance. This discrepancy between cells harboring wild-type or mutant EGFR possibly ascribe to variant cellular dependency for survival to EGFR pathways (48). The inhibitory effect of HJD+E combination surmounted the threshold that cancer cells could bear, and induced more lethal damage to cells with mutant EGFR.

STAT3 are important proto-carcinoma transcription factors in a variety of cancers (49–51). STAT3 activation has been reported in approximately 50% of NSCLC primary tumors and lung cancer-derived cell lines, which is one of the most important carcinogenic factors in NSCLC (52,53). STAT3 activation induces the transcription of a variety of genes (such as c-myc, cyclin D1, bcl-2) that play a key role in the pathogenesis of lung cancer (54). Previous findings have shown that EGFR mutations activate STAT3 (55). In addition, it has been shown that abnormal activation of STAT3 can be observed in a variety of drug-resistant cells, and inhibition of STAT3 activation can alleviate drug resistance (14,56,57). Erlotinib therapy resulted in the feedback activation of STAT3 in H1975 cells, and we later transfected HCC827, HCC827ER and H1975 cells with siRNAs. siSTAT3 significantly inhibited STAT3 and enhanced drug response. The result proved that STAT3 caused erlotinib resistance, in accordance with previous findings (58). In our study, HJD promoted erlotinib to enhance caspase 3 activation and tumor cell apoptosis. The combination of HJD and erlotinib blunted the expression of STAT3 and Bcl-2, suggesting HJD can inhibit the STAT3 signaling pathway to overcome TKI resistance.

In our study, HJD significantly alleviates resistance by regulating the STAT3/Bcl-2 signaling pathway. In addition, the results suggest that inhibition of the STAT3/Bcl-2 pathway contributed greatly to the abolishment of acquired resistance to erlotinib. Combination therapy of HJD and erlotinib offers a promising prospect for enhancing drug response and extending survival in patients with lung cancer.

Glossary

Abbreviations

SDC

Shuangdan capsule

EGFR

epidermal growth factor receptor

TKI

tyrosine kinase inhibitors

NSCLC

non-small cell lung cancer

HCC827ER

erlotinib-resistant HCC827 cell line

FBS

fetal bovine serum

Funding

Funding was provided by the Medical innovation team of Jiangsu province (CXTDB2017003), Jiangsu Province's colleges and universities (integration of Chinese and Western medicine).

Availability of data and materials

The datasets used during the present study are available from the corresponding author upon reasonable request.

Authors' contributions

RZ and LW designed and performed experiments. ZX and BL analyzed the data and wrote the manuscript. XZ and QN also performed the experiments. LZ analyzed the data. All authors read and approved the manuscript and agree to be accountable for all aspects of the research in ensuring that the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Ethics approval and consent to participate

The animal experiments were approved by the ethics committee of the Affiliated Hospital of Integrated Traditional Chinese and Western Medicine (Nanjing, China).

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no conflict of interest.