The A549 cell from NSCLC cell strain was provided by the School of Basic Medical Sciences, Zhejiang University. These epithelial-like cells are polygonal in shape with more regular dimensions and grow attached to a substrate in discrete patches.

The light-yellow EPP powder was provided by the School of Chemistry and Bioengineering, Guilin University of Technology, and DOX was from Zhejiang Provincial People’s Hospital. In addition, Roswell Park Memorial Institute (RPMI) medium (Cat# 11875-093), 0.25% trypsin (Cat# 25200-072), and penicillin-streptomycin glutamine (PSG) (Cat# 10378-016), and Fetal bovine serum (FBS) (Cat# A5669701) were purchased from Invitrogen Shanghai Trading Co., Ltd. (Gibco, Shanghai, China). Anhydrous ethanol (Cat# 64-17-5) was obtained from Sinopharm Chemical Reagent Co., Ltd. (Ourchem, Hangzhou, Zhejiang, China) and stored at 4 °C. The E-plate view 96 proliferation plate (Cat# 300601020) was purchased from Hangzhou Rongbang Biotechnology Co., Ltd. (Agilent, Hangzhou, Zhejiang, China). The ATP assay kit (Cat# 103592-100) and mitochondrial stress assay kit (Cat# 103015-100) were from Hangzhou BioSmile Biotechnology Co., Ltd. (Agilent, Hangzhou, Zhejiang, China). The six-well plates (Cat# 3799), centrifuge tubes (Cat# 430829), flow cytometry tubes (Cat# 352235), and 40 µm cell strainers (Cat# 352340) were purchased from Shanghai Pufei Biotechnology Co., Ltd. (Shanghai, China). The Annexin V-FITC/PI apoptosis detection kit (Cat# AP101) and PI cell cycle kit (Cat# CCS012) were from Hangzhou MultiSciences Biotechnology Co., Ltd. (MultiSciences, Hangzhou, Zhejiang, China)

First, 100 mg of EPP powder was taken and placed in a 50 mL centrifuge tube. Next, 10 mL of ddH₂O (0.22 µm) (Millipore, Hangzhou, Zhejiang, China) was slowly added to the tube and the powder was dissolved by ultrasonic apparatus (Xiaomeichaosheng, Kunshan, China). Then, an autoclave (Yamato, Chongqing, China) was used for sterilization and sterile ddH₂O was added to make a 10 mL solution. Finally, the 10 mg/mL EPP stock solution was made. Finally, 1 mg/mL DOX stock solution was made by dissolving 1 mg DOX powder into 1 mL of sterile saline.

The EPP group: the working concentrations of EPP were sequentially diluted in cell culture medium to 0.2, 0.4, 0.8, 1.6, and 3.2 mg/mL.

The DOX group: the working concentrations of DOX were sequentially diluted to 1, 2, 5, and 10 µg/mL in the cell culture medium.

The EPP+DOX group: DOX solutions at concentrations of 1, 2, and 5 µg/mL were mixed with EPP solutions at concentrations of 0.2, 0.4, 0.8, 1.6, and 3.2 mg/mL.

The A549 cell line of the human non-small cell lung cancer (NSCLC) were provided by the Prof. Yuehai Ke (originally purchased from ATCC). The cell line was maintained at 37 °C with 5% CO₂, validated by short tandem repeat(STR) profiling and tested negative for mycoplasma contamination using MycoSensor PCR assay kit (Agilent, Hangzhou, China). A549 cells were cultured in RPMI medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin glutamine (PSG). The medium was replaced every 1–2 days. When they were 80% confluent, the cells were passaged with 0.25% trypsin. This process was repeated for 5 passages.

The eSight system integrates real-time and label-free analysis technology with live cell imaging for cell phenotype analysis and cell-growth tracking. A suspension of 4 $\times$ 10³ cells/well was seeded in the E-Plate View 96 plate. The system measured the cell index (CI) value every 15 minutes. Cell images were captured using a 10 $\times$ objective lens at 3-hour intervals, with 4 images taken per well. This continuous monitoring lasted for 24 hours. The old medium of the control group was replaced with fresh culture medium with no drug. The culture medium of the experimental group was supplemented with different drugs: EPP, DOX, or the combination of the two, with 4 replicate wells for each group. The system would measure the CI value every 2 minutes and captured 4 images at 1-hour intervals. This continuous monitoring lasted for 72 hours. Then, the real-time dynamic curves of cell growth were plotted, and cell morphology was imaged to determine the optimal treatment duration. The half-inhibitory concentration (IC₅₀) was calculated to determine the optimal drug concentration.

A549 cells were cultured until they grow exponentially. After discarding the old culture medium, cultured cells with no drug administration were used as control groups. The cells used in experimental groups were treated with EPP, DOX, or the combination of both. Each group had 3 replicates. Next, the cells were cultured for another 24 hours. After trypsin digestion and centrifugation, the cell pellets were collected for flow cytometry to analyze the cell’s apoptosis and cell cycle.

(1) Cell Apoptosis Analysis: After the cell pellets were resuspended in 500 µL of 1$\times$ binding buffer, a single-cell suspension was obtained. 5 µL of Annexin V-FITC staining solution and 10 µL of PI staining solution were transferred to each tube. After mixing the solutions thoroughly, all the tubes were incubated on ice for 5 minutes in darkness. Then, the cell suspension was filtered by a cell strainer with 40 µm mesh. Next, it was transferred into the tubes of flow cytometry for further analysis.

(2) Cell Cycle Analysis: The cell pellets were washed and centrifuged in pre-chilled phosphate-buffered saline (PBS) for three times. After washing, the supernatant was discarded. The cell pellets were resuspended in 300 µL of pre-chilled PBS, followed by slow and uniform addition of 700 µL of pre-chilled absolute ethanol. After mixing, the fixed cells were kept at 4 °C overnight. The supernatant was then discarded, and the cell pellets were washed again using the pre-chilled PBS. The cells were resuspended in 500 µL of PBS to get a single-cell suspension. Next, 1 µL of RNase A (20 µg/mL) and 2.5 µL of PI staining solution (5 µg/mL) were added to the suspension. The mixture was incubated at room temperature for another 10 minutes in the darkness. Next, the cell suspension was filtered by a cell strainer, which had 40 µm mesh and was connected with the tubes of flow cytometry for further analysis.

A549 cells were cultured until they grow exponentially. After discarding the old culture medium, cultured cells with no drug administration were used as the control group. Those used in experimental groups were treated with EPP, DOX, or the EPP+DOX combination, respectively. At least 6 replicates were set up per condition. The cells were then cultured for another 24 hours. Before the experiment, the instrument and the probe plate were heated, and the probe plate was hydrated. When we started the experiment, we first prepared the analysis and detection solution to wash the cells. The ATP production rate assay kit and the mitochondrial stress test kit were used to measure the ATP production rate and the mitochondrial respiration and standard drugs was added to the probe plate. Then, the probe plate was mixed with the cell plate and placed in the instrument. After the measurement, 10 µL of radio immunoprecipitation assay lysis buffer (RIPA) was transferred into each well in the plate to lyse the cells. After two freeze-thaw cycles at –80 °C, we calculated the protein content in each well and the cellular ATP production rate to assess the mitochondrial respiration.

Representative results were selected from the experiment data for presentation. FlowJo_V10 software (BD Biosciences, San Jose, CA, USA), ModFit LT Version 5 software (Verity Software House, Topsham, ME, USA), and GraphPad Prism 9.0 software (GraphPad Software, La Jolla, CA, USA) were used to process the data. The values of IC₅₀ were reported as the mean $\pm$ standard deviation (SD). One-Way ANOVA was adopted to compare the means of independent groups to justify statistical evidence that the associated population means were significantly different. The sample size was set at n = 3. A p-value less than 0.05 was statistically significant.

Half-maximal inhibitory concentration (IC₅₀) is the most popular method to measure a drug’s efficacy. It indicates the necessary amount of drug to inhibit 50% cell growth—the lower the IC₅₀ value, the more potent the drug will be in anti-tumor activity [20].

3.1.1 Inhibition of A549 Cell Proliferation by EPP

A549 cells were plated for proliferate exponentially and then treated with EPP at different concentrations (0.2, 0.4, 0.8, 1.6, and 3.2 mg/mL). The eSight system was adopted to monitor the cells for 72 hours and draw the cell proliferation curves (Fig. 1) in real time. Compared with the control group (p 0.0001), the dose-dependent inhibition of A549 cell growth by EPP was observed. After 18 hours of monitoring, the proliferation curves of the low-dose group (0.2, 0.4, 0.8 mg/mL) increased, while the proliferation curves of the high-dose group (1.6, 3.2 mg/mL) reached a plateau. The proliferation curves of the low-dose group (0.2, 0.4, 0.8 mg/mL) decreased after 24 hours while the proliferation curves of the high-dose group (1.6, 3.2 mg/mL) decreased to the lowest point. After 48 hours, proliferation curves in the low-dose group (0.2, 0.4, 0.8 mg/mL) rapidly declined, while the decrease speed of high-dose group (1.6, 3.2 mg/mL) was much slower. These changes in cell proliferation curves indicate that EPP had an inhibitory effect on A549 cell proliferation. At 24 hours of drug exposure, EPP’s inhibition effects on A549 cell proliferation were all significant in various concentrations (p 0.0001). This suggests that 24 hours can be an optimal detection time for subsequent experiments. After 24 hours of treatment of EPP on A549 cells, the IC₅₀ reached 0.6 mg/mL. Therefore, 0.6 mg/mL of EPP was selected for the following experiments.

Fig. 1.

The effect of Enteromorpha prolifera polysaccaharide (EPP) on the proliferation of A549 cell. (A) The real-time dose-dependent curve of EPP. (B) The IC₅₀ of EPP. (C) The statistic of cell proliferation inhibition of EPP on A549 cell at various time points. ****p 0.0001, ^𝑛𝑠p $\mathrm{>}$ 0.05 (n = 4). IC₅₀, Half-maximal inhibitory concentration; ns, non-Significant.

3.1.2 DOX’s Inhibitory Effect on A549 Cell Proliferation

A549 cells were treated with DOX at the concentration of 1 µg/mL, 2 µg/mL, 5 µg/mL, and 10 µg/mL. The eSight system was used to monitor the treatment results and draw the cell proliferation curves. Compared with the control group (p 0.0001), after 24 hours of DOX treatment, there was a significant difference in cell viability (Fig. 2). Therefore, the concentration of DOX was key to the inhibitory effect. The IC₅₀ for DOX’s effect on A549 cells after 24 hours was calculated to be 2 µg/mL. Therefore, DOX at a concentration of 2 µg/mL was selected for subsequent experiments.

Fig. 2.

The effect of doxorubicin hydrochloride (DOX) on the proliferation of A549 cell. (A) The real-time dose-dependent curve of DOX. (B) The IC₅₀ of DOX. (C) The statistic of cell proliferation inhibition of DOX on A549 cell at various time points. ****p 0.0001 (n = 4).

3.1.3 Inhibitory Effect on A549 Cell Proliferation by the Mixture of EPP and DOX

In order to minimize toxic side effects and reduce activity in A549 cells, we need to optimize the concentration of EPP and DOX. This experiment compared the inhibitory effect on cell proliferation of the combined drug at different concentrations. Specifically, 1 µg/mL, 2 µg/mL, and 5 µg/mL of DOX and 0.2 µg/mL, 0.4 µg/mL, 0.8 µg/mL, 1.6 µg/mL, and 3.2 mg/mL of EPP were tested. A549 cells were treated with these mixtures for 72 hours. According to the results, the cell viability decreased in all groups (Fig. 3). When we mixed 2 µg/mL DOX with 0.4 mg/mL of EPP (p 0.0001), the most significant inhibitory effect was achieved. Therefore, the mixture of 0.4 mg/mL EPP and 2 µg/mL DOX increased the inhibitory effect of DOX on A549 lung cancer cells. Accordingly, the mixture was selected for subsequent experiments.

Fig. 3.

The effect of combination drug treatment of EPP and DOX on the proliferation of A549 cell. ****p 0.0001 vs. the Control group; ^𝑛𝑠p $\mathrm{>}$ 0.05 vs. the EPP and DOX groups (n = 4). ns, non-Significant.

The optimal concentration was found to effectively inhibit the proliferation of A549 cells after comparing the effects of EPP alone, DOX alone and the mixture of EPP and DOX. In addition, the best synergistic effect of EPP on DOX was 0.4 mg/mL, rather than 0.6 mg/mL, when EPP was used as a single liquid. Therefore, in the subsequent experiments, single EPP liquid was set at 0.6 mg/mL; single DOX liquid was set at 2 µg/mL; and the mixture of EPP and DOX was set at 0.4 mg/mL and 2 µg/mL respectively.

3.1.4 A549 Cell Morphology Imaging Results

Fig. 4 shows the cell morphology under the eSight system. In the control group, A549 cells had a spindle-shaped morphology and the cell density showed exponential growth overtime. After 72 hours of drug exposure, the cells shrank in volume. In the single EPP treatment group, compared with the control group, A549 cells began to shrink after 24 hours of treatment, indicating a certain inhibitory effect of EPP. In both the single DOX treatment group and the EPP+DOX mixture treatment group, after 24 hours of drug exposure, growth inhibition was also observed. A549 cell experienced shrinkage, cytoplasmic condensation, and gained darker color. In the mixture treatment group, the shrinkage of A549 cell membrane was even more pronounced. The cell became smaller with darker nucleus and more condensed cytoplasm. It color became darker than those in the single DOX treatment group. These observations were consistent with the curve of mixture treatment, which means that the mixture of EPP and DOX demonstrated a greater inhibitory effect on A549 cell proliferation.

Fig. 4.

The effect of EPP, DOX and combination drug treatment of EPP and DOX on the morphology of A549 cell. Scale bar: 100 µm.

The real-time energy metabolism analysis system (Seahorse XF 96) allows for real-time monitoring of cellular glycolysis and aerobic respiration, making it possible to analyze the intracellular parameters such as glycolytic, mitochondrial ATP production rate, and mitochondrial respiratory function in a dynamic way. This system provides a better understanding of the changes in cellular bioenergetics [24, 25]. Glycolysis is a set of reactions that converts glucose to pyruvate or lactate. It is a crucial energy source for tumor cells, the presence of can be a mark of cancer. ATP serves as the primary source of energy acquisition and storage in living organisms, while cellular apoptosis is an active cell death process that consumes ATP. Mitochondria plays a vital role in generating ATP within cells, and mitochondrial respiration is closely associated with cellular ATP level. Decreased mitochondrial function often coincides with a reduction in ATP level [26]. Therefore, inhibiting glycolysis to lower ATP level and impairing mitochondrial respiratory function can kill tumor cells targetedly.

3.3.1 Impact of Drugs on ATP Production Rate in A549 Cells

Fig. 7 shows the results of treating A549 cells with single EPP, single DOX, and the mixture of EPP and DOX for 24 hours. Compared with the control group, the EPP+DOX mixture treatment significantly inhibited glycolytic ATP production rate (p 0.01) and mitochondrial ATP production rate (p 0.0001). This indicates that ATP production can be reduced by the combination of EPP and DOX, thereby blocking the tumor cells’ proliferation.

Fig. 7.

The effect of combination drug treatment of EPP and DOX on the ATP production rate of A549 cell. ****p 0.0001, ***p 0.001, *p 0.05, ^𝑛𝑠p $>$ 0.05 (n = 6). glyco, glycogen; mito, mitochondria.

3.3.2 Impact of Drugs on Mitochondrial Respiratory Function in A549 Cells

Following the treatment of A549 cells with single EPP, single DOX, and the mixture of EPP and DOX for 24 hours (Fig. 8), it was observed that the EPP+DOX mixture treatment led to a significant decrease in ATP production (p 0.01) compared with the control group. This suggests that the combination of EPP and DOX will damage mitochondrial respiration in A549 cells, thereby inhibiting the energy production that supports tumor growth and survival. Consequently, tumor cell apoptosis occurs in an irreversible way.

Fig. 8.

The effect of combination drug treatment of EPP and DOX on the mitochondrial respiratory function of A549 cell. ****p 0.0001, ***p 0.001, ^𝑛𝑠p $>$ 0.05 (n = 6). OCR, Oxygen Consumption Rate.

Doxorubicin hydrochloride (DOX) is an antineoplastic chemotherapeutic drug under anthracycline class. It shows various anti-tumor activities and is widely used to treat lung cancer, breast cancer, and gastric cancer [23]. However, DOX has toxic side effect and tumors will get resistance to it. To improve its clinical use and avoid detrimental impact on patient, it is significant to find solutions.

Traditional Chinese medicine (TCM) is the result of Chinese culture and wisdom. Now, more and more TCM drugs are used to enhance the efficacy of therapies, reduce drug toxicity, inhibit tumor growth, and prevent drug resistance, especially in the field of cancer treatment [4]. Its active ingredients can increase tumor cells’ sensitivity to DOX, reduce drug resistance, control the growth of tumor cells and alleviate the adverse effect [4, 5, 27]. EPP, an acidic polysaccharide found in Enteromorpha, possesses unique anti-tumor and immunomodulatory properties [19, 28]. In this study, the mixture of EPP and DOX was used to treat A549 lung cancer cells. By examining cell proliferation, apoptosis, cell cycle distribution, mitochondrial function, among others, this study has probed into the inhibitory effect of the mixture on the activity of A549 lung cancer cells.

The eSight system is used to monitor the inhibiting effect of EPP, DOX and their mixture on A549 cells in real-time. Results showed that different concentrations of EPP and DOX could inhibit A549 cell growth that was dose independent. The calculated IC₅₀ values indicated that 0.6 mg/mL EPP and 2 µg/mL DOX can be used in the follow-up experiment to study the mixture’s inhibiting effect on A549 cell activity. Furthermore, from the analysis of the curves of a single treatment based on real-time monitoring and the observation of A549 cell morphology at different time points, significant differences were found between the treatment and the control group after 24 hours of drug exposure. These findings paved the way for evaluating the effect of drugs in future studies.

To better use pharmacological properties and improve the efficacy of DOX, this study mixed DOX with EPP to lower its toxic side effect of DOX while still inhibiting the growth of A549 lung cancer cells [29]. We used the eSight system (as shown in Fig. 3) to make the real-time proliferation curves that show the changes in A549 cells treated with different concentrations of EPP mixed with different concentrations of DOX. We observed that the low-dose EPP+DOX group induced cell apoptosis initially and, as time extended, cells were led to death, resulting in the inhibition of A549 cell activity. In comparison, the high-dose EPP+DOX group rapidly induced cell necrosis, achieving the inhibitory effect on A549 cell activity [30]. Compared with the control group, the treatment groups with a single drug, or the combination of EPP+DOX, show dose-dependent characteristics in terms of CI values and morphological changes in A549 cells. Specifically, the mixture of 0.4 mg/mL EPP and 2 µg/mL DOX (Fig. 9) had significantly higher inhibitory effect. These findings suggested that the synergistic effect of EPP and DOX substantially enhanced the inhibitory effect on A549 cell activity.

Fig. 9.

The effect of combination drug treatment of EPP and DOX on the cell proliferative activity of A549 cell. ****p 0.0001 (n = 4).