Lung cancer is one of the most common and most lethal malignancies in the world, and the effect of current treatment is severely limited1. Because of the poor development of the pulmonary sensory nerve, lung cancer is usually asymptomatic in the early and middle stages. Only when the tumor spreads to the pleura, some symptoms such as chest pain and hemoptysis will occur2. The patients already lose the chance of undergoing an operation when most of them are diagnosed at a late stage. There are many clinical cases in which Chinese herbal medicine has been used in the treatment of cancer as an auxiliary drug that has good curative effect and has been used in clinic.
Traditional Chinese medicine holds the view that the deficiency of vital energy is the premise of lung cancer3; the combination of abnormal change of qi, blood and body fluid, and the invasion of pathogenic factors leads to the pathologic process, which finally generates carcinoma of the lungs.
Shenqi Fuzheng (SQFZ) is used under the guidance of the theory of traditional Chinese medicine. Codonopsis pilosula and Astragalus membranaceus are the main raw materials of SQFZ, and the effective components are total saponins, astragaloside, and polysaccharides4. This formulation has been developed as an injection and is widely used in clinic, the effective components having the function of enhancing the immunity of the body. Combined with chemotherapy drugs, it can improve the efficacy, immune function, and quality of life of patients5.
For example, Hao and colleagues studied the efficacy and safety of the SQFZ-combined first-line chemotherapy for the treatment of non–small cell lung cancer using meta-analysis. A total of 43 randomized controlled trials were incorporated into the combined pharmacotherapy and monotherapy groups, with the result that the objective remission rate increased by about 20% compared with the control group [relative risk (RR)=1.23; 95% confidence interval (CI), 1.11–1.35; P<0.0001], the disease control rate increased by 11% (RR=1.11; 95% CI, 1.07–1.16; P<0.0001), the incidence of toxicity was about 50% lower than the control group (RR=0.59; 95% CI, 0.53–0.66; P<0.0001), and the immune function increased about 3.2 times (mean deviation=3.23; 95% CI, 2.86–3.60; P<0.00001)6.
To illustrate, the use of SQFZ injection combined with carboplatin chemotherapy in patients with gastrointestinal reaction and blood toxicity significantly reduced these symptoms compared with the control group7. One study of SQFZ injection treatment of patients with advanced non–small cell lung cancer found that CD4+ was significantly increased, and CD8+ obviously declined after treatment, showing that SQFZ injection can significantly improve the body’s immune funtion8.
Because of clinical orientation, the related research of SQFZ injection mainly focuses on enhancing the immune function of patients with chemotherapy; whether there is a direct effect on the tumor growth, metastasis and apoptosis is unknown, and there is very little research about whether SQFZ combined with chemotherapy drugs will affect the activity of chemotherapy drugs. On the basis of these, in this study, we combined SQFZ and docetaxel to explore the effect of their combination on lung cancer cells. Docetaxel is the first-line anticancer drug9,10 that is an analogue of paclitaxel. The main mechanism of docetaxel is inhibition of tumor cells by binding to tubular protein, promoting tubulin assembly into microtubules, while inhibiting its depolymerization, making cells stay in the G2/M phase11–13.
This study investigated the anticancer effect of SQFZ combined with docetaxel on the lung cancer cell lines, A549 and mouse lung cancer cell (LLC); it was found that SQFZ can significantly increase the growth inhibitory effect of low-concentration docetaxel on A549 and LLC cells from the cell morphologic integrity of the drug used; with regard to the effects of drugs on tumor migration ability and apoptosis of lung cancer cells, the combination of drugs had effects on cell morphology, migration, and apoptosis. Thus, the synergistic effect of SQFZ adjuvant chemotherapy was verified.
Materials and methods
The human lung cancer cell line A549 was presented by the Chinese People’s Liberation Army academy of military medical sciences, and the LLC line LLC was purchased from the Cell Library Committee of the Chinese Academy of Sciences (Shanghai, China). Both lung cancer cell lines were cultured using cell culture medium (Gibco) with 10% fetal bovine serum (Gibco) and 1% penicillin-streptomycin solution (Gibco) at 37°C with 5% CO2/95% air.
The cell viability of cells treated with SQFZ injection (Lizon Group Limin Pharmaceutical, Zhuhai, China) and docetaxel (Newbio Pharm-tech, Wuhan, China) was determined using the Thiazolan experiment (MTT) assay. A549 and LLC cells (100 μL/well) were plated in 96-well plates (Corning) at a density of 1×105 cell/mL and then incubated at 37°C for 24 hours. Thereafter, the culture medium was removed and replaced with a fresh medium with different concentrations of SQFZ and docetaxel. After incubation for 24 hours, 20 μL MTT (Amersco) solution (0.5 mg/mL) was added to each well, and the plates were incubated at 37°C until blue deposits were visible. Thereafter, the MTT solution was removed and 150 μL DMSO (Sigma) was added for a further incubation of 10 minutes. Absorbance was measured at 490 nm with a Multiskan Go microrplate reader (Thermo). Inhibition ratio was calculated with the equation:
Morphologic changes of cells
For the morphology, A549 and LLC cells were seeded into confocal culture dish at a density of 5×104 cell/mL. Each culture dish contains 2 mL cell suspension. After culturing for 24 hours, the culture medium was removed and replaced with a fresh medium with different concentrations of SQFZ and docetaxel. Then, after incubation for 24 hours, the cells were washed twice in phosphate buffer (PBS), and 4% paraformaldehyde was applied to fix for half an hour. Thereafter, cells were dyed with DAPI–Cell stain (Solarbio, Beijing) (1:100 1 mL) for 20 minutes and washed twice in PBS. The cells on the confocal culture dish were then dyed with DAPI (10 μg/mL, 100 μL) for 10 minutes; thereafter, the DAPI (Solarbio) was removed and replaced with Hank’s Balanced Salt Solution (Gibco) (1 mL). A confocal laser scanning microscope (FV1000; Olympus) was used to examine cell morphology.
Cell migration and invasion assays
The cell migration and invasion assay was performed using a 24-well Transwell (Corning) chamber. To the migration, A549 cells were trypsinized and resuspended at 5×104 cell/mL in 200 μL of serum-free medium, which contains different concentrations of SQFZ and docetaxel. Thereafter, the cells were seeded onto the upper chamber with a noncoated membrane. The bottom chamber was filled with 0.6 mL of cell culture medium with 10% fetal bovine serum. Twenty-four hours later, noninvading cells on the upper surface of the chamber were removed by cotton swabs, and the cells on the lower surface of filters were treated with 4% methanol (Beijing Dingguo Changsheng Biotechnology) for 30 minutes and stained with 0.1% crystal violet (KeyGEN BioTECH) for 30 minutes and then washed twice in 1% PBS. Images of A549 cells were captured using an inverted microscope at a magnification of ×100. For the invasion assay, A549 cells were collected and resuspended at a density of 1×105 cell/mL in serum-free medium, and seeded into the upper chamber with Matrigel-coated membrane. Other procedures were the same as migration assay. The number of cells was counted in 5 randomly selected fields. The Migration/Invasion inhibition rate was calculated as mentioned below:
Flow cytometry analyses
A549 cells were trysinized and resuspended at 1×105 cell/mL in 2 mL medium of each well at 6-well plates; after incubation for 24 hours at 37°C, the cells were treated with different concentrations of SQFZ and docetaxel for 24 hours; thereafter, the cells were trypsinized and washed in buffer once and centrifuged at 260g for 5 minutes. The supernatant was discarded, and the cells were resuspended in 200 μL Annexin V binding buffer, 5 μL FITC-Annexin V, and 5 μL PI (Solarbio). After incubation in the dark for 15 minutes, the cells were harvested to analyze the released fluorescence, using FACSCalibur flow cytometre (BD). The number of necrotic, living, early apoptotic, and late apoptotic or dead cells detected was characterized by dot plots.
Statistics and data analysis
The data were analyzed by the Wilcoxon Txo-Sample test (SAS 9.3). *P<0.05 compared with docetaxel alone group, **P<0.01 compared with docetaxel alone group.
Cell growth inhibition by SQFZ and docetaxel
The MTT assay showed that using SQFZ with 5 concentrations (100, 50, 10, 1, 0.1 mL/L ) alone had no difference compared with the control group. Therefore, it proves that SQFZ has no inhibition effect on 2 kinds of cells. The result of adding docetaxel with 9 concentrations (0.08, 0.8, 8, 40, 80, 800, 8000, 40000, 80000 μg/L) alone to A549 and LLC cells is shown in Figure 1. Obviously, the growth of A549 and LLC cells was inhibited by an increasing amount of docetaxel added. In addition, the inhibition effect of 5 low concentrations of docetaxel alone and combined with 1 and 10 mL/L SQFZ is shown in Figure 2.
Compared with docetaxel alone, the inhibitory effect of docetaxel combined with SQFZ on A549 and LLC cells was enhanced. In addition, the inhibition rate increased with the increasing concentration of SQFZ. As regards combination use, its effect on A549 and LLC cells is more obvious at a low concentration of docetaxel; to sum it up, when the concentration of docetaxel is 0.8 μg/L, it has the highest combination effect.
Morphologic changes of cells
Cell morphology reflects the effect of drugs on cell anatomy. DAPI–Cell stain can dye the cell membrane red, and DAPI can dye the nuclei blue. The result of SQFZ used alone is shown in Figure 3. Using SQFZ with 3 concentrations alone makes no difference compared with the control group, and the cells grew well. The result of Docetaxel used alone and combined with SQFZ is shown in Figure 4. When docetaxel is used alone, with increasing concentrations, the cell morphology was gradually destroyed, but, when combined with a concentration of 0.1 mL/L SQFZ and low concentration of docetaxel in the cell membrane, the nucleus was obviously damaged. This result is similar to MTT; at low concentrations of docetaxel, SQFZ’s effect is more obvious; it has the highest combination effect when the concentration of docetaxel is 80 μg/L.
In addition, we also compared the effects of different concentrations of SQFZ combined with docetaxel (Fig. 5), as shown in the figure; the 3 concentrations of SQFZ had no influence on the cell morphology. In addition, because SQFZ need to administrate in advance of docetaxel in clinic, we also did the experiment that gave SQFZ 24 hours advance of docetaxel. However, there is no obvious difference in morphology. Cell morphology showed obvious changes after the use of docetaxel combined with SQFZ, including the nucleus and cell membrane rupture. LLC cells also showed similar results (Figs. 6–8).
Effect of SQFZ and docetaxel on A549 cell migration and invasion
As shown in Figure 9, A549 cells were treated with 6 concentrations of SQFZ; all of them had obvious migration inhibition rate. The concentration of 80 μg/L of docetaxel showing a high inhibition rate may be caused by cell death. However, when docetaxel was combined with SQFZ, the migration inhibition rate increased obviously (Fig. 10). When the concentration of docetaxel is 0.08 μg/L, it has the highest combination effect. Similar to the migration, SQFZ also has a significant effect on inhibiting cell invasion (Fig. 11). The invasion inhibition rate is about 50%; in the ability to resist cell invasion, the 2 drugs showed a good combination effect (Fig. 12). At the concentration of 0.08 μg/L docetaxel, it has a significant combination effect.
Docetaxel combined with SQFZ-induced apoptosis in A549 cells
The rate of apoptosis is expressed as the percentage of early apoptosis (Q4) + the percentage of late apoptosis (Q2). SQFZ still does not inhibit lung cancer cells (Fig. 13), but, after combination with docetaxel, the apoptosis rate was increased obviously (Fig. 14). There is also a synergistic effect on lung cancer cell apoptosis. When the concentration of docetaxel is 0.8 μg/L, it has the highest combination effect. In addition, it did not have an effect on the apoptosis rate of A549 cells with SQFZ delivery in advance. For the SQFZ administration in the advance group, with the increase of SQFZ concentration, the apoptosis rate increased slightly, which may be caused by residual amounts of SQFZ (Fig. 15).
At present, SQFZ injection showed good results as an adjunct therapy to treat lung cancer, esophagus cancer, and colon cancer, but the direct mechanism of SQFZ on cancer cells is still unclear14–16. Clinical reports suggest that chemotherapeutic agents combined with SQFZ could enhance the immunity of patients and reduce side effects, and also could increase the sensitivity of the tumor to chemotherapeutic drugs.
Through experimental study, it is proved that SQFZ alone has no direct effect on human lung cancer cell line A549 and LLC line LLC. However, after being combined with docetaxel, it can significantly enhance the effect of docetaxel on cell growth inhibition, morphologic change, and cell migration inhibition, and promotes apoptosis in lung cancer cells. First, the inhibition effect of SQFZ and docetaxel on cell growth was studied by the MTT method. The results showed that SQFZ could significantly enhance the inhibitory rate of docetaxel on A549 and LLC cells in a certain range of microconcentrations (0.08–8 μg/L). In general, it has a most significant synergistic effect when docetaxel is at a concentration of 0.8 μg/L and is combined with SQFZ. Docetaxel is usually used once every 3 weeks, with a dose of 75 mg/m2. According to the relevant literature of Pharmacokinetic study results of docetaxel17, 24 hours after administration, the plasma concentration was under 15 μg/L, and the decrease in concentration can make it difficult to play a direct antitumor effect. According to the results of cell study, at the dosing interval of docetaxel, the use of SQFZ can improve the tumor inhibitory rate, prolong the duration of the drug, and enhance clinical efficacy. As reported in the literature, Liao et al18 compared the effect of the combination of chemotherapy with SQFZ and monotherapy on the therapeutic effect on 80 patients with gastric cancer. After treatment, the cancer embryo antigen of the combined group was significantly reduced as well as different levels of CD3+, CD4+, and CD4+/CD8+ increased. SQFZ has a sensitization effect on cisplatin resistance of A549 cells; after the administration of SQFZ, it can inhibit the expression of drug resistance–related genes, MDR1 and LRP, in A549 cells, thereby enhancing the sensitivity of cells to cisplatin19,20.
The result on cell morphology is similar to MTT; at a low concentration of docetaxel, the docetaxel alone and docetaxel combined with SQFZ was compared. From the picture of cell morphology after administration of SQFZ and docetaxel. The nucleus and cell membrane changed significantly, the death of cells was increased, and the cell rupture was obvious. This may be related to the enhancement of the sensitivity of chemotherapeutic drugs, which is expressed in the form of cell morphology. The third is the cell migration and invasion experiment; it is reported that the extracts of A. membranaceus and C. pilosula can inhibit the migration of vascular endothelial cells induced by lung cancer cells, which may be related to the inhibition of Vascular endothelial growth factor expression in lung cancer cells21. A range of concentrations (0.01–10 mL/L) of SQFZ could inhibit the migration of lung cancer cells significantly, and the inhibition rate was about 50%; it can also significantly increase the migration rate of docetaxel after combination; SQFZ can increase the inhibited migration by about 40% compared with that of the low concentration docetaxel; as regards the high migration inhibition of the high concentration of docetaxel, it may be due to too much cell death, but for invasion inhibition, the synergic effect was not prominent.
Some studies indicate that the sensitization effect of SQFZ may be related to the promotion of expression of apoptosis genes22. Some traditional Chinese medicines such as Center-Supplementing and Qi-Boosting Decoction can active the PI3K/Akt signal pathway, down regulate P-glycoprotein (P-gp), multidrug resistance–associated protein (MRP), and lung resistance–related protein (LRP)23,24. In the apoptosis experiment, SQFZ can also enhance the apoptosis effect of docetaxel, and the most significant synergy effect was shown at docetaxel (0.8 μg/L) combining with SQFZ. However, there was no difference between giving SQFZ 24 hours in advance and giving both medicine at the same time, which may be due to the different mechanism of the body’s level and the cellular level.
In this experiment, the effect of SQFZ combined with docetaxel on lung cancer was demonstrated visually and concretely, and the experiment proves that the anticancer effect of docetaxel in low concentrations on lung cancer cells can be increased using SQFZ injection. Hence, the treatment effect of using a low concentration of docetaxel together with SQFZ injection can also be comparable to the effect of high concentration docetaxel but with lower side effect. It provides a basis for clinical practice. In terms of toxicity, allergic reaction could be the common adverse reaction when using combined pharmacotherapy of SQFZ25. Predictably, for patients with lung cancer who have lost their chances of surgery, the combination of SQFZ is a preferred treatment.
The program was approved by the Ethics Committee of Beijing University of Chinese Medicine.
Sources of funding
Supported by Beijing technology development project (No. 2017110031009880); the preliminary study on the antilung cancer of compound liposome of docetaxel and Shenqi Fuzheng (No. 2017-JYB-XS-067).
C.W. and B.Y. contributed equally to this work. All authors read and approved the manuscript.
Conflict of interest disclosures
The authors declare that they have no financial conflict of interest with regard to the content of this report.
Research registration unique identifying number (UIN)
The authors thank Yang Lu for insightful discussions and Beijing University of Chinese Medicine for laboratory support.
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