Oridonin (Ori) with a purity of up to 98% was purchased from Shanghai Yuanye Bio-Technology Co., Ltd. Ori was dissolved in DMSO (Sigma) at a stock solution of 100 mM and stored at −20°C. Docetaxel (DTX) was provided by Shanghai Jinhe Bio-Technology Co., Ltd. (Shanghai, China).

Human NSCLC cell lines A549 and NCI-H1975 (H1975) were obtained from American Type Culture Collection (ATCC). A549 cells were cultured in Dulbecco modified Eagle medium (DMEM). H1975 cells were cultured in RPMI-1640 medium. DMEM and RPMI-1640 medium were supplemented with 10% fetal bovine serum (FBS) (Hyclone), 100 U/ml penicillin, and 100 μg/ml streptomycin and cultured in a humidified atmosphere with 5% CO₂ at 37°C.

Cells were seeded into 96-well plate and pre-cultured for 24 h, then treated with Ori for 24 h. Cell cytotoxicity was determined by MTT assay. The absorbance was measured at 570 nm by Automated Microplated Reader (Bio-Tek, Winooski, VT, USA), and the cell death rate was calculated as follows: Inhibition Rate (%) = (average A₅₇₀ of the control group − average A₅₇₀ of the experimental group)/(average A₅₇₀ of the control group − average A₅₇₀ of the blank group) ×100%. Cell viability was estimated by trypan blue dye exclusion (28).

Cells were suspended in 1 ml of RPMI-1640 containing 0.3% low-melting-point agarose (Amresco, Solon, OH, USA) and 10% FBS, and plated on a bottom layer containing 0.6% agarose and 10% FBS in 6-well plate in triplicate. After 2 weeks, plates were stained with 0.2% gentian violet and the colonies were counted under a light microscope (29).

An invasion assay was carried out using a 24-well plate (Corning). A polyvinyl-pyrrolidone-free poly-carbonate filter (8 μm pore size) (Corning) was coated with Matrigel (BD Biosciences). The lower chamber was filled with medium containing 20% FBS as chemoattractant. The coated filter and upper chamber were laid over the lower chamber. Cells (1×10⁴ cells/well) were preincubated with Ori for 30 min at room temperature, and then cell suspension containing Ori was seeded onto the upper chamber wells. After incubation for 20 h at 37°C, the filter was fixed and stained with 2% ethanol containing 0.2% crystal violet (15 min). After being dried, the stained cells were enumerated under a light microscope at ×10 objective. For quantification, the invaded stained cells on the other side of the membrane were extracted with 33% acetic acid. The absorbance of the eluted stain was determined at 570 nm.

Cells (4×10⁵ cells/2 ml) were seeded in a 6-well plate and incubated at 37°C until 90 to 100% confluence. After the confluent cells were scratched with a 200 μl pipette tip, followed by washing with PBS, and then treated with Ori in a complete medium. After 24 h of incubation, the cells were fixed and stained with 2% ethanol containing 0.2% crystal violet powder (15 min), and randomly chosen fields were photographed under a light microscope at ×4 objective. The number of cells migrated into the scratched area was calculated.

Cells (5×10⁴ cells/well) preincubated with Ori for 30 min at 37°C were seeded in a 96-well plate coated with matrigel for 20 min at 37°C. Unattached cells were removed by washing with PBS. Attached cells were fixed in 4% paraformaldehyde for 15 min, stained with 0.02% crystal violet solution for 10 min, and randomly chosen fields were photographed under a Leica DMI 400B microscope. Then, to quantify the number of attached cells, crystal violet was dissolved with 70% ethanol and OD was measured by micro-plate reader at 570 nm, reference 405 nm. The adhesion cells were calculated as a percentage of adhesion.

Cell pellets were lysed in RIPA buffer containing 50 mM Tris pH 8.0, 150 mM NaCl, 0.1% SDS, 0.5% deoxycholate, 1% NP-40, 1 mM DTT, 1 mM NaF, 1 mM sodium vanadate, 1 mM PMSF (Sigma), and 1% protease inhibitors cocktail (Merck). Protein extracts were quantitated and loaded on 8 to 12% sodium dodecyl sulfate polyacrylamide gel, electrophoresed, and transferred to a PVDF membrane (Millipore). The membrane was incubated with primary antibody, washed, and incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (Pierce). Detection was performed by using a chemiluminescent Western detection kit (Cell Signaling Technology). The antibodies used were anti-CIP2A, anti-ERK2, anti-phospho-Akt (Ser473), anti-Akt (Santa Cruz Biotechnology), anti-phospho-ERK1/2 (Thr202/Tyr204), anti-EGFR (L858R), anti-phospho-EGFR (Y1173), anti-PP2Ac (Cell Signaling Technology), anti-MMP-12 (Proteintech), anti-MMP-2 (Epitomics), and anti-GAPDH (Sangon Biotech Co., Ltd., AB10016) antibodies.

H1975 cells were seeded in 6-well plates and allowed to grow to 80% confluency. The cells were then maintained in serum-free medium for 12 h prior to designated treatments with Ori for 24 h. Conditioned medium was then collected, cleared and mixed with 5X SDS loading buffer, and subjected to electrophoresis on a 10% SDS-PAGE gel containing 0.1% gelatin. After electrophoresis, the gels were washed in renaturing buffer (pH 7.5, 2.5% Triton X-100) for 30 min, 4 times, and equilibrated in developing buffer (50 mM Tris-HCl pH 7.5, 10 mM CaCl₂, and 1 mM ZnCl₂) for 30 min and finally incubated in fresh developing buffer at 37°C for 24 h to allow digestion of the gelatin. The gelatinolytic activity of MMPs was visualized by staining the gels with 0.5% Coomassie blue R-250 in 45% methanol, 10% acetic acid and destained with 45% methanol, 10% acetic acid until clear bands suggestive of gelatin digestion appeared.

The total RNA of the cells was isolated using the TRIzol Reagent (Invitrogen) and the phenol-chloroform extraction method according to the manufacturer's instruction. Total RNA (2 μg) was annealed with random primers at 65°C for 5 min. The cDNA was synthesized using a 1st-STRAND cDNA Synthesis kit (Fermentas). For PCR amplification, primers are as follows: GAPDH, forward primer: 5′-TCACCAGGGCTGCTTTTA-3′, reverse primer: 5′-AAGGTCATCCCTGAGCTGAA-3′; MMP-12 forward primer: 5′-TTGTTCCTCACTGCTGTTCAC-3′; reverse primer: 5′-GTCCATCATCTGTCTCCTTTC-3′.

H1975 cells were seeded in 12-well culture plates. At a confluency of 70%, cells were transfected with the pAP-1-Luc plasmids (Beyotime Institute of Biotechnology) using Lipofectamine 3000 (Invitrogen) according to manufacturer's instruction. After transfection for 4 h, the cells were treated with Ori for 20 h. Firefly luciferase activities were assayed using the Luciferase Assay System (Promega) according to the manufacturer's instructions.

H1975 cells treated with Ori for 24 h were processed for ChIP assay using a ChIP-IT^® Express Enzymatic Shearing kit (Active Motif). Briefly, immunoprecipitation was performed with anti-c-Fos (component of AP-1, Cell Signaling Technology) or rabbit IgG as a control. The AP-1 binding site of MMP-12 promoter was detected by real-time PCR using the following primers: 5′-GCTAATTGATCCATTGT-3′ (forward) and 5′-TCTAGCCTAAGTTCC-3′ (reverse).

PP2A immunoprecipitation phosphatase assay kit (Upstate Biotechnology, Temecula, CA, USA) was used to measure phosphate release as an index of phosphatase activity according to the manufacturer's instructions. Briefly, 100 μg of protein isolated from cells was incubated with 4 μg anti-PP2A monoclonal antibody overnight. 40 μl of protein A agarose beads were added and the mixture was incubated at 4°C for 2 h. Subsequently, the beads were collected and washed three times with 700 μl of ice-cold TBS and one time with 500 μl of Ser/Thr Assay buffer. The beads were further incubated with 750 mM phosphopeptide in assay buffer for 10 min at 30°C with constant agitation. Malachite Green Phosphate Detection Solution (100 μl) was added and the absorbance at 650 nm was measured on a microplate reader (30).

Drug combination is widely used in cancer treatment to achieve synergistic therapeutic effect and overcome drug resistance in clinic. To estimate the effect of Ori and DTX combination, the combination index (CI) was calculated by the Chou-Talalay equation (29). H1975 cells were seeded in 96-well plates. Drugs were added alone or together at indicated concentration. The inhibition effect was measured by MTT assay as mentioned above. The formula of CI = (D) Ori/(Dx) Ori + (D)DTX/(Dx)DTX ((D)Ori and (D)DTX: the doses of compounds Ori and DTX, respectively, necessary to produce the same effect in combination. Dx: the dose of one compound alone required producing an effect). With this formula and assistance of CalcuSyn software (version 2.1), the combined effects of the two compounds can be assessed as follows: CI<1 indicates synergism; CI=1 indicates additive effect; and CI>1 indicates antagonism.

Equal number of female and male nude immunodeficient mice (nu/nu), 6–8 weeks old, were purchased from Hunan SJA Laboratory Animal Co., Ltd., and maintained and monitored in a specific pathogen-free environment. All animal studies were conducted according to protocols approved by the Hubei University of Medicine Animal Care and Use Committee, complying with the rules of Regulations for the Administration of Affairs Concerning Ex-Perimental Animals (Approved by the State Council of China). The mice were injected subcutaneously with NSCLC H1975 cells (2.5×10⁶) suspended in 100 μl RPMI-1640 medium into the right flank of each mouse. Treatments were started when the tumors reached a palpable size. Mice were randomly divided into three groups and treated with Ori (30 mg/kg, n=8), Gefitinib (30 mg/kg, n=7) or vehicle control (n=7) for 3 weeks. Caliper measurements of the longest perpendicular tumor diameters were performed twice a week to estimate the tumor volume, using the following formula: 4π/3 × (width/2)² × (length/2), representing the 3-dimensional volume of an ellipse. Animals were sacrificed when tumors reached 1.5 cm or if the mice appeared moribund to prevent unnecessary morbidity to the mice. At the time of sacrifice, tumors were excised for immunohistochemistry.

For malin-fixed, paraffin-embedded tissues from mice were selected for immunohistochemical examination by using an indirect immunoperoxidase method. The antibodies used for immunohistochemical staining were pEGFR, CIP2A, and MMP-12.

All experiments were repeated at least three times and the data are presented as the mean ± SD unless noted otherwise. Differences between data groups were evaluated for significance using Student t-test of unpaired data or one way analysis of variance and Bonferroni post-test. P-values <0.05 indicate statistical significance.

A549 and H1975 cells were seeded in 96-well plates for 24 h and then treated with different Ori concentrations (Fig. 1B and C). After 24 h, cell viability was evaluated through an MTT assay in accordance with the manufacturer's protocol. The absorbance at 570 nm was determined by using an automated microplate reader. Ori exerted moderate cytotoxicity against the A549 and H1975 cells with IC₅₀ of 55.91 and 15.53 μM, respectively (Table I). Trypan blue exclusion assay revealed that Ori rapidly reduced the number of viable H1975 cells (Fig. 1D) in a dose- and time-dependent manner. We also investigated the effect of Ori on cell colony formation and found that Ori significantly inhibited the clonogenic ability of H1975 (Fig. 1E). These results suggested that Ori inhibited the anchorage-dependent (cell proliferation) and anchorage-independent (colony formation) growth of H1975 cells.

We determined whether Ori inhibited the invasive behavior of H1975 cells. An invasion assay was performed using Matrigel-coated 24-well microchemotaxis chambers in the presence of Ori. Fig. 2A shows that Ori (0–20 μM) markedly suppressed H1975 cell invasion. To explore the effect of Ori on migration, we treated the H1975 cells with Ori (0–20 μM) and determined cell migration after 24 h. Fig. 2B shows that Ori significantly and dose-dependently reduced H1975 cell migration. We evaluated the effect of Ori on cell adhesion. Fig. 2C illustrates that Ori also inhibited the adhesion of H1975 cells onto the Matrigel in a concentration-dependent manner compared with the untreated control cells. These results suggested that Ori exerted an anti-invasive behavior toward lung cancer at moderate concentrations.

EGFR and extracellular regulated protein kinases (ERK) play important roles in the proliferation and invasion of NSCLC cells (31). Ori can dose-dependently reduce the phosphorylation levels of EGFR and ERK without causing evident changes in the total EGFR and total ERK expression levels in H1975 cells (Fig. 3A and B). Considering that MMP-12, which can be regulated by ERK1/2 pathway, plays an important role in the migration and invasion of cancer cells (32), we examined the expression and gelatinolytic activity of MMP-12 in H1975 cells exposed to different Ori concentrations (Fig. 3C and D). The results showed that the gelatinolytic activity and expression levels of MMP-12 were significantly reduced in the Ori-treated H1975 cells in a dose-dependent manner compared with the control groups. AP-1, which is regulated by MAPKs, such as ERK, JNK, and P38 kinase, is a critical transcription factor that activates MMP-12 transcription (33). We also examined the effect of Ori on the transcriptional activity of AP-1 through luciferase reporter assay and found that 15 and 20 μM of Ori significantly inhibited AP-1 transcription activity (Fig. 3E). Chromatin immunoprecipitation (ChIP) assays also revealed that 15 μM of Ori inhibited the DNA binding of c-Fos (component of AP-1) to MMP-12 promoter (Fig. 3F). These results indicated that Ori can significantly suppress the invasive behavior (invasion, migration, and adhesion) of H1975 cells via the EGFR/ERK/AP-1/MMP-12 signaling pathway.

The Akt pathway controls tumor cell growth, survival, progression, apoptosis, invasion, and metastasis, and Akt is a critical locus of cancer multidrug resistance and fragility (34). Ori selectively targets the Akt signaling pathway and inhibits apoptosis of cervical carcinoma HeLa cell line (35). Therefore, we investigated the effect of Ori on Akt phosphorylation in H1975 cells. Our results showed that the phosphorylation level of Akt was dose-dependently downregulated, and the total Akt level did not evidently change in the Ori-treated H1975 cells compared with the control group (Fig. 4A). Furthermore, we determined the changes in the expression of PP2A, which is an upstream phosphatase of Akt. The results suggested that Ori could upregulate the PP2Ac (catalytic subunit) expression level. To elucidate the mechanism of Ori-induced PP2Ac upregulation, we examined the PP2A activity and found that this activity was upregulated in the Ori-treated H1975 cells compared with that in the control group, which is possibly one of the reasons leading to the Ori-mediated downregulation of Akt phosphorylation (Fig. 4B). Moreover, CIP2A is an endogenous inhibitory protein of PP2A, which demonstrates cancer-promoting profile in NSCLC (21). We subsequently detected the CIP2A expression and found that Ori significantly downregulated the expression of the CIP2A protein (Fig. 4C). Thus, Ori elicits an inhibitory effect on H1975 cells by regulating the CIP2A/PP2A/Akt signal cascade.

On the basis of these results, we concluded that Ori inhibited gefitinib-resistant NSCLC growth and cell invasion by inhibiting the EGFR/ERK/AP-1/MMP-12 signaling pathway and the CIP2A/PP2A/Akt signal cascade (Fig. 4D).

One or more drugs are commonly used in clinics to improve the therapeutic efficacy and reduce the side effects on patients and non-target tissues. Furthermore, an optimal combined treatment may reduce or postpone drug resistance. Microtubular-targeted chemotherapy agents, such as DTX, are among the most widely prescribed first- and second-line chemotherapeutic options for patients suffering from common malignancies, including lung cancer (36). Unfortunately, these malignancies usually develop primary resistance to DTX; thus, drug resistance poses an important clinical problem. We examined whether the combination of Ori and DTX synergistically inhibits the NSCLC treatment. We further chose the low-toxicity (2.5 and 5 μM) doses of Ori and DTX (4 and 8 nM) to detect (Fig. 5A). The combination of Ori and DTX elicited a greater effect on the H1975 cells than the sole treatment of DTX or Ori (P<0.05). We also analyzed the combination index (CI) by using a formula assisted by CalcuSyn software (version 2.1) and found that the CIs were <1 (Fig. 5B, Table II). This finding indicated that Ori and DTX synergistically affected the H1975 cells. Western blot analysis further confirmed this synergistic effect and revealed that the combination of Ori and DTX reduced the expression levels of pEGFR, pERK, and MMP-12 (Fig. 5C). This combination also influenced the CIP2A/PP2A/Akt signal cascade (Fig. 5D).

To determine the antitumor effect of Ori on NSCLC in vivo, we subcutaneously inoculated 2.5×10⁶ H1975 cells in 100 μl of RPMI-1640 medium in the right flank of nude mice to generate xenografted murine models. Once the tumors grew to a measurable size, each group was administered with the vehicle control, gefitinib (30 mg/kg), and Ori (30 mg/kg) 5 times per week for 21 days. The tumor-bearing mice were humanely sacrificed when their tumors reached 1.5 cm in diameter or when paralysis or major compromise in their quality of life occurred. We found that Ori efficiently repressed tumor growth compared with the vehicle control or gefitinib (P<0.01; Fig. 6A and B). Ori treatment also significantly reduced the tumor weight of the mice (Fig. 6C). In addition, Ori treatment did not significantly reduce the body weight of the mice. This finding suggested that Ori did not cause evident side effects (Fig. 6D). All of the mice were euthanized, and the tumor specimens were examined through immunohistochemistry; the results showed that the expression levels of p-EGFR, MMP-12, and CIP2A were downregulated in the Ori-treated groups (Fig. 6E). Therefore, Ori could be a potential therapeutic agent against gefitinib-resistant NSCLC.