# Alcohol Extract of YFXJ Decoction Reverses the Drug Resistance of Human Lung Adenocarcinoma Cell Line A549/DDP

Authors: Zhongliang Liu; Yaping Ding; Yizhou Tian; Da Yu
Journal: Computational and Mathematical Methods in Medicine
 (2022)

---

## Abstract

Objective. Lung cancer is the wide and common tumor. This study was designed to explore the effect of YFXJ formula on non-small-cell lung cancer (NSCLC) cell lines. Methods. YFXJ formula (mainly composed of Astragalus membranaceus, Atractylodes macrocephala, Radix Saposhnikoviae, Radix Glehniae, coix seed, Herba Sabina chinensis, Hedyotis diffusa, Pericarpium Citri Reticulatae, sarcophagus martensii, Prunella vulgaris, Meretrix meretrix, and oyster) was extracted with 75% ethanol. We performed MTT, FACS, TUNEL, and mass spectrometry to study the effect of YFXJ formula on A549/DDP cells. Results. The results showed that YFXJ could inhibit the growth of A549/DDP cells, and it can reverse the sensitivity of A549/DDP cells to cisplatin. YFXJ inhibits the expression of MDR1, MRP1, and LRP genes in A549/DDP cells. Conclusion. YFXJ formula can reverse the drug resistance of A549/DDP cell, which could be through activation of autophagy.


---

## Body

## 1. Introduction

Lung cancer is the wide and common tumor and causes of human death. There are about 1.5 million people died due to this disease every year. Non-small-cell lung cancer (NSCLC) accounts for nearly 85% of newly diagnosed lung cancer every year, such as adenocarcinoma, squamous cell carcinoma, and large cell lung cancer [1]. Stage II NSCLC can be cured by surgery. However, 85%-90% of patients with NSCLC have lost the best opportunity for surgery [2]. At present, radiotherapy and chemotherapy are the conventional treatment for advanced non-small-cell lung cancer, especially the emergence of new second-generation (pemetrexed disodium) and third-generation chemotherapy drugs (docetaxel, paclitaxel, vinorelbine, etc.) has significantly improved the efficacy of chemotherapy. In addition, the emergence of immunotherapy, interventional therapy, and other means further improves the efficiency of tumor treatment and prolongs the incidence of cancer. However, biological immunotherapies are not widely used due to the limitations of price and efficacy. This makes chemotherapy become the main force of antitumor treatment. Changing chemotherapy resistance can benefit patients to a greater extent and more widely, which is very important for prolonging the survival of patients. Therefore, how to delay, reverse, or partially reverse chemotherapy resistance is particularly important, very necessary, and of practical significance.In the 1970s and 1980s, cisplatin, carboplatin, ifosfamide, vinblastine, vinpocetine, mitomycin, epirubicin, and etoposide were considered as effective drugs for NSCLC chemotherapy. When used in single drug chemotherapy, the effective rate was at least 15%. When combined with cisplatin, the effective rate was 30%, and the 1-year survival rate was 10% ~12%. It can be seen that single chemotherapy and combined chemotherapy have low efficiency and short survival rate. The economic cost of the combination of two third-generation chemotherapy drugs or the combination of the third-generation drugs and targeted therapy drugs is generally difficult for most people to bear. Due to historical habits and other reasons, platinum containing chemotherapy is widely used. However, there is an important bottleneck in the treatment of chemotherapeutic drugs, namely, acquired drug resistance, especially the change of drug resistance of tumor cells after chemotherapy, which limits the efficacy of chemotherapy regimens and affects the further improvement of clinical efficacy of patients.YFXJ formula (mainly composed of Astragalus membranaceus, Atractylodes macrocephala, Radix Saposhnikoviae, Radix Glehniae, Coix seed, Herba Sabina chinensis, Hedyotis diffusa, Pericarpium Citri Reticulatae, sarcophagus martensii, Prunella vulgaris, Meretrix meretrix, and oyster) is used to cure cancers. Previous experiments have shown that YFXJ formula can prolong the disease-free progression time and delay drug resistance. Therefore, further in vitro experiments to clarify the reversion and antitumor effect of YFXJ formula on drug-resistant cells naturally become our further research direction.Here, we prepared the alcohol extract of YFXJ formula and analyzed it by MTT, FACS, TUNEL, and mass spectrometry to study the effect of YFXJ formula on NSCLC cell line. Our data suggest that the alcohol extract of YFXJ decoction can reverse the drug resistance of A549/DDP cells.


## 2. Material and Methods

### 2.1. Preparation of Alcohol Extract of YFXJ Formula

According to the formula of YFXJ (Astragalus membranaceus 30 g, Fangfeng 10 g, Atractylodes macrocephala 30 g, scorpion 3 g, Nansha ginseng 15 g, shijianchuan 30 g), weigh a certain amount of decoctions of medicinal materials, add 8 times the volume of 75% ethanol, reflux, extract, and decoct for 2 times, once 1 hour. The decoctions were combined, centrifuged, and dried to obtain an ethanolic extract. Before the experiment, an appropriate amount of dry powder of the ethanol extract was weighed and dissolved in the medium by vortexing, ultrasonic wave, water bath, and other methods. Filters are used to filter and prepare solutions of the desired concentration. The solution was stored at 4°C until use.


### 2.2. Cell Lines and Grouping

A549 cell line and human lung cancer multidrug resistance cell line A549/DPP were purchased from the Institute of Biochemistry and Cell Biology (Shanghai Academy of life sciences). The sensitive of A549 cells was resuscitated and cultured. The logarithmic growth phase cells were divided into experimental group (A549/DDP + conventional medium + YFXJ decoction alcohol extract), control group 1 (A549/DDP + conventional medium), and control group 2 (sensitive cell line A549 + conventional medium). All three groups were added with cisplatin for drug intervention.


### 2.3. MTT Method

The MTT method was used to determine the effect of alcohol extract of YFXJ formula on A549/DDP cells at 24, 48, 72, and 96 h, and the sensitive cell line A549 was used as control. The effect of YFXJ formula on A549/DDP cell line was assessed by IC50 value. IC50 was calculated by fitting inhibition curve with GraphPad Prism 5.0 software:reversalmultipleRF=IC50controlgroup/IC50 (YFXJ treatment group).


### 2.4. Flow Cytometry and TUNEL

A549/DDP cells were cultured overnight in starvation. After treated with different concentrations of alcohol extract of YFXJ formula, the cells were cultured in incubators of 37°C, 5% CO2, and saturated humidity for 12, 24, 36, and 48 h. The cell cycle and apoptosis were measured using flow cytometry and TUNEL, respectively.


### 2.5. Mass Spectrometry (ICP-MS)

Alcohol extract of YFXJ formula was used to treat A549/DDP cells for 36 h and then further treated with 10 g/ml DDP for 4 h. After being centrifugated for 10 min at 14000 RPM/min, the supernatant was vacuum dried for 24 h, and 70μL PBS solution was added, diluted and mixed, washed repeatedly, and completely transferred to a 40 ml beaker. At the same time, high-grade pure nitric acid (2 ml) was added to the electric heating plate for digestion and finally adds high-grade pure perchloric acid (1 ml) to digest the sample at high temperature. After cooling, the sample was put into 10 ml volumetric flask. The sample was filtered with filter paper before measurement, and then the accumulation concentration of DDP in cells was detected by ICP-MS.


### 2.6. Statistical Analysis

GraphPad Prism 5.0 software, SPSS18.0 statistical software,t-test, and F test were used for quantitative treatment and analysis. Specific statistical methods may be used when some experimental steps are involved, which is closely related to the computer software used.


## 2.1. Preparation of Alcohol Extract of YFXJ Formula

According to the formula of YFXJ (Astragalus membranaceus 30 g, Fangfeng 10 g, Atractylodes macrocephala 30 g, scorpion 3 g, Nansha ginseng 15 g, shijianchuan 30 g), weigh a certain amount of decoctions of medicinal materials, add 8 times the volume of 75% ethanol, reflux, extract, and decoct for 2 times, once 1 hour. The decoctions were combined, centrifuged, and dried to obtain an ethanolic extract. Before the experiment, an appropriate amount of dry powder of the ethanol extract was weighed and dissolved in the medium by vortexing, ultrasonic wave, water bath, and other methods. Filters are used to filter and prepare solutions of the desired concentration. The solution was stored at 4°C until use.


## 2.2. Cell Lines and Grouping

A549 cell line and human lung cancer multidrug resistance cell line A549/DPP were purchased from the Institute of Biochemistry and Cell Biology (Shanghai Academy of life sciences). The sensitive of A549 cells was resuscitated and cultured. The logarithmic growth phase cells were divided into experimental group (A549/DDP + conventional medium + YFXJ decoction alcohol extract), control group 1 (A549/DDP + conventional medium), and control group 2 (sensitive cell line A549 + conventional medium). All three groups were added with cisplatin for drug intervention.


## 2.3. MTT Method

The MTT method was used to determine the effect of alcohol extract of YFXJ formula on A549/DDP cells at 24, 48, 72, and 96 h, and the sensitive cell line A549 was used as control. The effect of YFXJ formula on A549/DDP cell line was assessed by IC50 value. IC50 was calculated by fitting inhibition curve with GraphPad Prism 5.0 software:reversalmultipleRF=IC50controlgroup/IC50 (YFXJ treatment group).


## 2.4. Flow Cytometry and TUNEL

A549/DDP cells were cultured overnight in starvation. After treated with different concentrations of alcohol extract of YFXJ formula, the cells were cultured in incubators of 37°C, 5% CO2, and saturated humidity for 12, 24, 36, and 48 h. The cell cycle and apoptosis were measured using flow cytometry and TUNEL, respectively.


## 2.5. Mass Spectrometry (ICP-MS)

Alcohol extract of YFXJ formula was used to treat A549/DDP cells for 36 h and then further treated with 10 g/ml DDP for 4 h. After being centrifugated for 10 min at 14000 RPM/min, the supernatant was vacuum dried for 24 h, and 70μL PBS solution was added, diluted and mixed, washed repeatedly, and completely transferred to a 40 ml beaker. At the same time, high-grade pure nitric acid (2 ml) was added to the electric heating plate for digestion and finally adds high-grade pure perchloric acid (1 ml) to digest the sample at high temperature. After cooling, the sample was put into 10 ml volumetric flask. The sample was filtered with filter paper before measurement, and then the accumulation concentration of DDP in cells was detected by ICP-MS.


## 2.6. Statistical Analysis

GraphPad Prism 5.0 software, SPSS18.0 statistical software,t-test, and F test were used for quantitative treatment and analysis. Specific statistical methods may be used when some experimental steps are involved, which is closely related to the computer software used.


## 3. Results

### 3.1. MTT Was Used to Detect the Effect of YFXJ on A549/DDP Cell Proliferation

MTT assay was used to study the effect of YFXJ on A549/DDP cells proliferation. After 48 h, the inhibition rates of YFXJ at 2 and 5μmol/L on A549/DDP cell proliferation were 4.97% and 14.71%, respectively (Figure 1). Therefore, when YFXJ concentration was 2, 5 μmol/L, it could be considered as nontoxic concentration (inhibitionrate<5%) and low toxicity concentration (inhibitionrate<15%), respectively. With these two concentrations, it could be considered that the growth rate of A549/DDP did not change obviously than that of the control group.Figure 1
Effect of YFXJ on the inhibition rate of A549/DDP cells YFXJ leads to growth inhibition of A549/DDP cells.∗P<0.05; ∗∗P<0.01.
(a)(b)


### 3.2. Effect of YFXJ on the A549/DPP Cell Sensibility for Cisplatin

The effects of A549/DPP cells on the sensitivity of A549/DPP cells to cisplatin were observed and detected when the concentration of YFXJ was 2μmol/L and 5 μmol/L, respectively. 0, 2, 5, 10, 20, 50, and 100 μ mol/L cisplatin was added into the corresponding holes. The four groups are as follows: A549 + cisplatin control group, A549/DPP + cisplatin group, A549/DPP + cisplatin +2 μmol/L YFXJ group, and A549/DPP + cisplatin +5 μmol/L YFXJ group. Then, CCK-8 assay was conducted to calculate the inhibition rate of the drug on the cells. Abscissa was the concentration of cisplatin and ordinate was inhibition rate.The inhibition rate of cisplatin on A549/DPP cell proliferation under the effect of 2 or 5μmol/L YFXJ was higher compared with the A549/DPP + cisplatin group, showing that YFXJ can increase the sensitivity of A549/DPP cells to cisplatin (Figure 2). According to the IC50 value, the IC50 of DDP alone on A549/DPP cells was 60.24; the IC50 of DDP + YFXJ 2 μmol/L on A549/DPP cells was 30.71; the IC50 of DDP + YFXJ 5 μmol/L on A549/DPP cells was 16.12. Therefore, the reversal multiples of 2 and 5 μ mol/L YFXJ were 1.96 and 3.74 times, respectively.Figure 2
YFXJ reverses the sensitivity of A549/DDP cells to cisplatin. YFXJ reverses the sensitivity of A549/DDP cells to cisplatin, compared with the control group,∗P<0.05.
(a)(b)


### 3.3. Effect of YFXJ Combined with Cisplatin on Apoptosis of A549/DPP Cells

Results was shown in Figure3, and the effect of apoptosis was detected by flow cytometry. The apoptosis rates of A549/DPP cells in the cisplatin group and 2 and 5 μmol/L YFXJ combined with the cisplatin group were higher compared with the control group (P<0.01). At the same time, YFXJ combined with the cisplatin group was better compared with the cisplatin alone group (P<0.01), and 5 μ mol/L YFXJ combined with cisplatin group was the best (P<0.01). It is suggested that YFXJ combined with cisplatin can induce A549/DDP cells apoptosis in a concentration-dependent manner.Figure 3
Effect of YFXJ combined with cisplatin on apoptosis of A549/DDP cells. YFXJ increased the apoptosis of A549/DDP cells to cisplatin, compared with the control group,∗P<0.05, ∗P<0.01.
(a)(b)(c)(d)(e)


### 3.4. Effect of YFXJ on MDR1, MRP1, and LRP Gene mRNA Expression in A549/DPP Cells

Next, we examined the effect of YFXJ on the mRNA expression of MDR1, MRP1, and LRP genes in A549/DPP cells. The mRNA levels of MDR1, MRP1, and LRP genes in A549/DPP cells decreased after YFXJ treatment. The MDR1 expression in the 2 and 5μmol/L group was 81.1% and 68.0% of the control group, the MRP1 mRNA expression was 82.1% and 73.9% of the control group, and the LRP mRNA expression was 70.3% and 63.3% of the control group, respectively. It shows that YFXJ can regulate the transcription of drug-resistant related genes (Figure 4). WB confirmed that the protein levels of MDR1, MRP1, and LRP genes in A549/DPP cells were decreased after YFXJ treatment (Figure 5). MDR1, MRP1, and LRP protein expression was downregulated by YFXJ in a dose-dependent manner.Figure 4
Effect of YFXJ on mRNA expression of MDR1, MRP1, and LRP genes in A549/DDP cells. YFXJ inhibits the mRNA expression of MDR1, MRP1, and LRP genes in A549/DDP cells.∗P<0.05; ∗∗P<0.01.Figure 5
Effect of YFXJ on the expression of MDR1, MRP1, and LRP proteins in A549/DDP cells. YFXJ inhibits the protein expression of MDR1, MRP1, and LRP genes in A549/DDP cells.∗P<0.05; ∗∗P<0.01.
(a)(b)


### 3.5. Effects of Different Concentrations of YFXJ on mRNA Expression of Autophagy-Related Gene in A549/DDP Cells

The mRNA of Akt, p53, PTEN, mTOR, ATG5, and LC3 genes which related to autophagy in A549/DPP cells showed a concentration dependent change after different YFXJ treatments. However, only at 50μmol/L concentration, YFXJ could significantly increase the expression of Akt (P<0.05, Figure 6). Together, YFXJ might reverse the drug resistance of A549/DDP through activation of autophagy.Figure 6
Effects of different concentrations of YFXJ on the mRNA expression of autophagy-related gene in A549/DDP cells.∗P<0.05; ∗∗P<0.01.


### 3.6. Effect of YFXJ on Autophagy-Related Molecular Protein Expression in Lung Cancer A549/DPP Cells

The results of WB showed that Akt, p53, PTEN, mTOR, ATG5, and LC3 related to autophagy in A549/DPP cells were obviously inhibited by YFXJ in a dose-dependent manner (Figure7). These results suggest that YFXJ can activate autophagy and promote A549/DPP cells apoptosis. Combined with the results of quantitative PCR, it is suggested that YFXJ may not directly inhibit the transcription level of mRNA but may affect the translation or degradation of protein.Figure 7
The effect of different concentrations of YFXJ on the expression of autophagy-related proteins in A549/DDP cells.


## 3.1. MTT Was Used to Detect the Effect of YFXJ on A549/DDP Cell Proliferation

MTT assay was used to study the effect of YFXJ on A549/DDP cells proliferation. After 48 h, the inhibition rates of YFXJ at 2 and 5μmol/L on A549/DDP cell proliferation were 4.97% and 14.71%, respectively (Figure 1). Therefore, when YFXJ concentration was 2, 5 μmol/L, it could be considered as nontoxic concentration (inhibitionrate<5%) and low toxicity concentration (inhibitionrate<15%), respectively. With these two concentrations, it could be considered that the growth rate of A549/DDP did not change obviously than that of the control group.Figure 1
Effect of YFXJ on the inhibition rate of A549/DDP cells YFXJ leads to growth inhibition of A549/DDP cells.∗P<0.05; ∗∗P<0.01.
(a)(b)


## 3.2. Effect of YFXJ on the A549/DPP Cell Sensibility for Cisplatin

The effects of A549/DPP cells on the sensitivity of A549/DPP cells to cisplatin were observed and detected when the concentration of YFXJ was 2μmol/L and 5 μmol/L, respectively. 0, 2, 5, 10, 20, 50, and 100 μ mol/L cisplatin was added into the corresponding holes. The four groups are as follows: A549 + cisplatin control group, A549/DPP + cisplatin group, A549/DPP + cisplatin +2 μmol/L YFXJ group, and A549/DPP + cisplatin +5 μmol/L YFXJ group. Then, CCK-8 assay was conducted to calculate the inhibition rate of the drug on the cells. Abscissa was the concentration of cisplatin and ordinate was inhibition rate.The inhibition rate of cisplatin on A549/DPP cell proliferation under the effect of 2 or 5μmol/L YFXJ was higher compared with the A549/DPP + cisplatin group, showing that YFXJ can increase the sensitivity of A549/DPP cells to cisplatin (Figure 2). According to the IC50 value, the IC50 of DDP alone on A549/DPP cells was 60.24; the IC50 of DDP + YFXJ 2 μmol/L on A549/DPP cells was 30.71; the IC50 of DDP + YFXJ 5 μmol/L on A549/DPP cells was 16.12. Therefore, the reversal multiples of 2 and 5 μ mol/L YFXJ were 1.96 and 3.74 times, respectively.Figure 2
YFXJ reverses the sensitivity of A549/DDP cells to cisplatin. YFXJ reverses the sensitivity of A549/DDP cells to cisplatin, compared with the control group,∗P<0.05.
(a)(b)


## 3.3. Effect of YFXJ Combined with Cisplatin on Apoptosis of A549/DPP Cells

Results was shown in Figure3, and the effect of apoptosis was detected by flow cytometry. The apoptosis rates of A549/DPP cells in the cisplatin group and 2 and 5 μmol/L YFXJ combined with the cisplatin group were higher compared with the control group (P<0.01). At the same time, YFXJ combined with the cisplatin group was better compared with the cisplatin alone group (P<0.01), and 5 μ mol/L YFXJ combined with cisplatin group was the best (P<0.01). It is suggested that YFXJ combined with cisplatin can induce A549/DDP cells apoptosis in a concentration-dependent manner.Figure 3
Effect of YFXJ combined with cisplatin on apoptosis of A549/DDP cells. YFXJ increased the apoptosis of A549/DDP cells to cisplatin, compared with the control group,∗P<0.05, ∗P<0.01.
(a)(b)(c)(d)(e)


## 3.4. Effect of YFXJ on MDR1, MRP1, and LRP Gene mRNA Expression in A549/DPP Cells

Next, we examined the effect of YFXJ on the mRNA expression of MDR1, MRP1, and LRP genes in A549/DPP cells. The mRNA levels of MDR1, MRP1, and LRP genes in A549/DPP cells decreased after YFXJ treatment. The MDR1 expression in the 2 and 5μmol/L group was 81.1% and 68.0% of the control group, the MRP1 mRNA expression was 82.1% and 73.9% of the control group, and the LRP mRNA expression was 70.3% and 63.3% of the control group, respectively. It shows that YFXJ can regulate the transcription of drug-resistant related genes (Figure 4). WB confirmed that the protein levels of MDR1, MRP1, and LRP genes in A549/DPP cells were decreased after YFXJ treatment (Figure 5). MDR1, MRP1, and LRP protein expression was downregulated by YFXJ in a dose-dependent manner.Figure 4
Effect of YFXJ on mRNA expression of MDR1, MRP1, and LRP genes in A549/DDP cells. YFXJ inhibits the mRNA expression of MDR1, MRP1, and LRP genes in A549/DDP cells.∗P<0.05; ∗∗P<0.01.Figure 5
Effect of YFXJ on the expression of MDR1, MRP1, and LRP proteins in A549/DDP cells. YFXJ inhibits the protein expression of MDR1, MRP1, and LRP genes in A549/DDP cells.∗P<0.05; ∗∗P<0.01.
(a)(b)


## 3.5. Effects of Different Concentrations of YFXJ on mRNA Expression of Autophagy-Related Gene in A549/DDP Cells

The mRNA of Akt, p53, PTEN, mTOR, ATG5, and LC3 genes which related to autophagy in A549/DPP cells showed a concentration dependent change after different YFXJ treatments. However, only at 50μmol/L concentration, YFXJ could significantly increase the expression of Akt (P<0.05, Figure 6). Together, YFXJ might reverse the drug resistance of A549/DDP through activation of autophagy.Figure 6
Effects of different concentrations of YFXJ on the mRNA expression of autophagy-related gene in A549/DDP cells.∗P<0.05; ∗∗P<0.01.


## 3.6. Effect of YFXJ on Autophagy-Related Molecular Protein Expression in Lung Cancer A549/DPP Cells

The results of WB showed that Akt, p53, PTEN, mTOR, ATG5, and LC3 related to autophagy in A549/DPP cells were obviously inhibited by YFXJ in a dose-dependent manner (Figure7). These results suggest that YFXJ can activate autophagy and promote A549/DPP cells apoptosis. Combined with the results of quantitative PCR, it is suggested that YFXJ may not directly inhibit the transcription level of mRNA but may affect the translation or degradation of protein.Figure 7
The effect of different concentrations of YFXJ on the expression of autophagy-related proteins in A549/DDP cells.


## 4. Discussion

Previous experiments have shown that YFXJ formula can prolong the disease-free progression time and delay drug resistance. Therefore, furtherin vitro experiments to clarify the reversion and antitumor effect of YFXJ formula on drug-resistant cells naturally become our further research direction.Targeted drugs further prolong the survival time of chemotherapy-resistant patients to a certain extent. With the emergence of new chemotherapy drugs, many clinical researches have shown that [3–9] for patients with advanced NSCLC with good prognosis factors, the combination of new chemotherapy drugs is better than single drug chemotherapy. In addition, meta-analysis showed that the two drug regimen was better than the single drug regimen in improving the survival time of patients, while the three drug regimen had increased adverse reactions and no significant improvement in survival time compared with the two drug regimen [10]. Compared with standard chemotherapy, the progression free time was prolonged by 2-4 months with fewer side effects, for example, EGFR inhibitors such as gefitinib, erlotinib, icotinib, and afatinib, ALK inhibitors such as clotriminib, and c-Met inhibitor tivantinib. However, due to the high cost, the cost of targeted drug treatment for half a year is more than 100000-150000. Moreover, drug resistance is inevitable, and some drugs are in the experimental stage, which leads to the limited universality of targeted drugs and bottleneck in improving the efficacy.Autophagy is a process of cellular metabolism and some organelles renew. It is that phagocytizing cytoplasmic proteins or organelles were enveloped into vesicles and then fused with lysosomes to degrade their contents. However, whether the role of autophagy is positive or negative has not been completely elucidated, especially in cancer research, which deserves attention. In this study, YFXJ activated autophagy to reverses the drug resistance of A549/DDP cells.Taken together, our results submit a theoretical basis for the influence of YFXJ recipe in the therapy of NSCLC and provide evidence support for further promoting the use of YFXJ formula. Moreover, Oyster and Meretrix are common raw materials of marine traditional Chinese medicine in Zhoushan area. This study can promote and improve the value of marine Chinese medicine and market economy.



---

*Source: 9089365-2022-07-21.xml*