Ophiopogonin B-inducing Pyroptosis Through Caspase-1/gsdmd Pathway Contributes to Alleviation of Paclitaxel Resistance in Lung Cancer Cells

Background: Drug resistance has become the main reason for the failure of tumor chemotherapy. In our previous study, ophiopogonin B (OP-B) has been veried to inhibit cell proliferation in numerous non-small cell lung cancer (NSCLC) cells. However, it is still unknown whether it can improve the drug resistance of lung cancer cells. Herein, we compared the inhibition effects of OP-B on NCI-H460, A549, A549/DDP and A549/PTX cells, and tried to nd out the most sensible cell line to OP-B and the underlying reasons. Methods: The sensitivity of NCI-H460, A549, A549/DDP, and A549/PTX cells to OP-B was determined by CCK-8 assay, and the results were further veried in orthotopic tumor nude mice model and zebrash tumor model. To identify pyroptosis in the cells, electron microscopy was used to observe cell morphology, ow cytometry was used to detect the mitochondrial membrane potential, and the LDH release rate was analyzed by microplate reader. Otherwise, immunouorescence and immunohistochemical staining assay, western blot and qRT-PCR were used for detection of pyroptosis-correlated pathway. Results: In vitro, A549/DDP cell was veried to be most sensitive to OP-B than NCI-H460, A549, or A549/PTX cells. In vivo, OP-B inhibited the growth of A549/DDP orthotopic tumor more signicantly than that of A549 both in nude mice and zebrash models. Cell morphological feature, mitochondrial membrane potential, LDH release rate, production of IL-1β and expression of Caspase-1/GSDMD all showed that pyroptosis happened more signicantly in A549/DDP cells than that in A549 cells after OP-B treatment. Conclusion: Though inducing more signicantly pyroptosis by activating Caspase-1/GSDMD pathway, OP-B relieved DDP resistance of A549 cells.


Introduction
In recent years, lung cancer has become the third leading cause of cancer-related diseases (1). The clinical classi cation of lung cancer is complex and diverse, while NSCLC accounts for up to 85% of the patients (2). Currently, chemotherapy remains the standard of treatment for NSCLC patients due to approximately 85-90% NSCLC having no driver mutations de ned for drug target (3). However, with the long-term application of basic chemotherapy drugs in clinical practice, the problem of tumor drug resistance becomes increasingly serious, which has been the main reason for the ine ciency of tumor chemotherapy (4). Therefore, it's very urgent to develop new drugs or nd sensitizers for them to improve clinical e cacy.
The mechanism of drug resistance in tumors are complex, which mainly includes drug concentration reduction in tumor cells, abnormal drug metabolic pathway, DNA damage repair dysfunction, regulation of autophagy, blocking of apoptotic pathways, and self-renewal or proliferation of tumor stem cells.
Pyroptosis is a type of programmed cell death that associated with in ammation, which characterized by cell swelling, destruction of membrane structural integrity and cytoplasmic contents release, like interleukin-1β (IL-1β), interleukin-18 (IL-18) and lactate dehydrogenase (LDH) (5). The activation of classical and non-classical pathway is separately dependent on Caspase-1 or Caspase-4/5/11 (6,7). Activated Caspase-1/4/5/11 catalyze GSDMD to cleave into GSDMD-N and GSDMD-C fragments, and GSDMD-N can bind and form pores at cell membrane, so as to lead pyroptosis and release IL-1β and IL-18 to extracellular environment (8,9). Shi J et al. found that GSDMD-N itself can directly induce pyroptosis, which indicates that GSDMD cleavage is really an important index to detect whether pyroptosis occurs or not (10).
Currently, some studies found that there is any connection between tumor resistance and pyroptosis (11).
For example, Wu M et al. combined BI2536 (an inhibitor of PLK1) with cisplatin (DDP) to treat ESCC cells and found the combination of two drugs had a synergistic effect on tumor inhibition and resulted in pyroptotic death of cancer cells by Caspase-3/GSDME pathway. Their further research revealed that BI2536 can reverse DDP resistance of ESCC by inhibiting DNA damage repair and inducing pyroptosis (12).
Traditional Chinese medicine has been used for thousands of years in China and has unique advantages in cancer treatment. Ophiopogonin B (OP-B) is a bioactive component extracted from Radix Ophiopogon Japonicus, a traditional Chinese medicine often used in pulmonary disease treatment. In our previous studies, OP-B had been veri ed to have signi cant inhibition effects on various NSCLC cells (13)(14)(15). Herein, we focused on comparing the sensitivity of DDP-resistant A549 (A549/DDP) and A549 cell lines to OP-B, and found that A549/DDP cells were more sensitive to OP-B, and the underlying mechanism may due to the induction of pyroptosis through Caspase-1/GSDMD pathway. Cell culture A549 and NCI-H460 cells were obtained from the Stem Cell Bank, Chinese Academy of Sciences (Shanghai, China). A549/DDP and A549/PTX cells were kind gifts from Professor Zhigang Guo (NanJing Normal University). A549, NCI-H460 cells were cultured in DMEM/F12 medium (Gibco, Australia), and A549/DDP, A549/PTX cells were cultured in RPMI 1640 medium (Gibco, Australia) with 10% fetal bovine serum (FBS; Gibco, Australia), supplemented with 1% penicillin/streptomycin solution (Gibco, Australia).

Materials And Methods
All of the cells were maintained at 37 °C in humidi ed atmosphere of 5% CO 2 .

Western blot analysis
The cells were lysed in radioimmunoprecipitation assay (RIPA) buffer (Beyotime, Shanghai, China) containing 1% phenylmethanesulfonyl uoride (PMSF) before suspended in sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) sample loading buffer (Beyotime, Shanghai, China), then separated on 12% SDS-PAGE (Beyotime, Shanghai, China) and transferred onto polyvinylidene uoride (PVDF) membranes (Thermo Fisher, US). After the membranes were blocked with 5% non-fat milk, they were incubated at 4 °C overnight with primary antibodies against Caspase-1 (1:500, CST, US), GSDMD Total RNA was extracted from A549 or A549/DDP cells using TRIzol reagent (Sangon Biotech) according to the manufacturer's protocol. Then, the RNA was reverse transcribed to cDNA using PrimeScript™ RT reagent Kit with gDNA Eraser (Takara). Quantitative real-time PCR was performed using cDNA primers speci c for mRNA. The gene GAPDH was used as an internal control. All the real-time PCR reactions were performed using Takara′s SYBR Premix Ex Taq™ II (Tli RNaseH Plus) in Applied Biosystems 7500 Fast Real-Time PCR System (Applied Biosystems). The 2 -△△Ct method was used for quanti cation and fold change for target genes was normalized by internal control.
When frozen sections were used for immuno uorescence, the sections were rst blocked with goat serum; the rest of the procedure was the same as that for the goat serum-blocked cells.

LDH Release Assay
The activity of LDH released into cell culture supernatants was detected using the CytoTox 96 Non-Radioactive Cytotoxicity Assay Kit (Omega) according to the manufacturer's protocol for analyzing pyroptosis.
In vivo xenograft assay (nude mice models and xenograft zebra sh) The BALB/c nude mice (4-week old) were maintained under speci c pathogen-free (SPF) conditions. Animal welfare and experimental procedures were performed in compliance with the National Institutes of Health Guidelines for the care and use of laboratory animals, and all protocols were approved by the Ethics Review Committee of Nanjing University of Chinese Medicine. To establish the orthotopic xenograft lung cancer model, the luciferase-expressing A549 or A549/DDP cell line with lentivirus was established, then A549 or A549/DDP cells (2×10 7 in 0.2ml medium of a 1:1 mixture of RPMI 1640 and Matrigel 354,248) were injected into right lung parenchyma of the mice, and the volume of tumors were monitored by luciferase imaging of live animals using an IVIS Spectrum bioluminescence imaging system (PerkinElmer, US) after intraperitoneal injection of 200 μl D-Luciferin substrate (15 mg/ml in DPBS, PerkinElmer). And the mice were mainly used to test the toxicity and pharmacological activity of OP-B on A549 or A549/DDP xenograft mice. The mice for toxicity-detection were divided into 5 groups (6 in each group), including polo-188 group (62.5mg/ml Poloxamer), OP-B groups (1.5 or 3mg/Kg OP-B), and the Normal or Mock group (saline). And the mice for pharmacological activity detection were divided into 9 groups, including the Normal group (saline), A549 or A549/DDP Mock groups (saline), A549 OP-Btreatment groups (1.5 or 3mg/Kg OP-B), A549/DDP OP-B-treatment groups (1.5 or 3mg/Kg OP-B), and A549 or A549/DDP cyclophosphamide groups (20mg/Kg cyclophosphamide). All of the mice were treated with intraperitoneal injection (i.p. daily, n=28). The polo-188 was formulated with 0.9% NaCl, and OP-B was formulated with polo-188. 25 days later, all mice' hearts, livers, lungs and kidneys were harvested, then half of the tissues were used for GPT, GOT and CRE detectinon with microplate test kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China), and the rest of them were used for haematoxylin-eosin (H&E) staining, Transmission Electron Microscope (TEM) observation, or immunohistochemistry and immuno uorescence observation.
AB/wt zebra sh embryos were raised at 28 °C in sh water. At 48 hours fertilization (hpf), A549 cells, labeled with a red uorescent dye for cell viability (Cell Tracker™ CM-DiI, Invitrogen, CA, USA) and resuspended in HBSS were injected into the yolk sac of zebra sh embryos (200 cells/embryo). Then, embryos were incubated at 34 °C. At 72 h post injection, the proliferation of tumor cells was evaluated through a uorescence stereomicroscope (OLYMPUS U-HGLGPSD, equipped with Cell Sens Entry software, Tokyo, Japan). The software Image J was used to quantify the proliferation rate of tumor cells.
The protocols for the animal experiments were approved by the Ethics Review Committee of Nanjing University of Chinese Medicine.
Transmission Electron Microscope (TEM) assay The model and OP-B groups were selected for TEM analysis to observe the morphology of A549 cells and A549/DDP cells. The mice were sacri ced, 1/3 of tissue was removed from lung and xed with 3% glutaraldehyde for more than 2 hours, then immobilized with 1% osmium acid for 2 hours. The tissues were dehydrated step by step with ethanol and acetone and then soaked overnight and embedding agent. After the tissues were embedded, polymerized, repaired, sliced and double stained with uranium acetate and lead citrate, they were observed by TEM JEM-1011 .

Statistical analysis
Data entry and all analyses were performed in a blinded fashion. All statistical analyses were performed using GraphPad Prism7.0 software. Statistical signi cance was calculated using two-tailed unpaired ttest on two experimental conditions or two-way ANOVA when repeated measures were compared, with p < 0.05 considered statistically signi cant. All graphs show mean values ± SEM.

Results
OP-B inhibited A549/DDP cell proliferation more signi cantly than that on A549 cells First of all, we used CCK-8 assay to test cell viability of A549, A549/PTX or A549/DDP cells under different doses of PTX and DDP for 24h to prove the drug resistance of A549/DDP and A549/PTX cells (Fig. 1b). Under the treatment of PTX, the IC 50 values of A549 and A549/PTX cells were 16 Next, we compared the inhibitory effects of OP-B on NCI-H460, A549, A549/PTX and A549/DDP cells, with a concentration range of 1.25~20μM for 24 or 48h treatment (Fig. 1c). The results suggested that among the four kinds of cells, A549/DDP was more sensitive to OP-B, the effective concentration was 2.5μM. More importantly, further combined action of OP-B and DDP (160μM) on A549 and A549/DDP cells for 24h veri ed that they had synergistic effect (Fig. 1d). Taken together, OP-B not only had signi cant inhibition effect on A549/DDP cells, but also increased the sensitivity of A549/DDP cells to DDP.
In order to determine whether OP-B has the same effect in vivo, we established the orthotopic xenograft lung cancer model and administrated the mice with 1.5mg/kg or 3mg/kg of OP-B for 25 consecutive days. The results showed that under the treatment of OP-B, mice weight between A549 and A549/DDP groups was basically the same (Fig. 1e). Meanwhile, the tumor volume of both A549 and A549/DDP groups decreased in dose and time dependent manner under the treatment of OP-B ( Fig. 1f and g), and the inhibition rate on A549/DDP group was more signi cant than that on A549 (Fig. 1h).
Inhibitory effect of OP-B on A549/DDP xenografted tumors in mice and zebra sh In order to verify whether OP-B has the same effect on tumor in situ, we inoculated A549 and A549/DDP cells on the right lung lobe of BALB/c nude mice, and monitor the tumor growth by bioluminescence image. After the models being successfully built, 3mg/kg of OP-B was administered to the mice consecutively for 14 days, meanwhile, bioluminescence images were taken on 0 and 14th days (Fig. 2a). Statistical results showed that the uorescence intensity in A549 and A549/DDP tumor-in-situ was signi cantly decreased by OP-B treatment (Fig. 2b and c), while between A549 and A549/DDP groups, OP-B inhibited A549/DDP tumor-in-situ more signi cantly than that in A549 groups (Fig. 2d).
In addition, we veri ed the effect of OP-B on proliferation of A549 and A549/DDP cells in zebra sh. To establish tumor model, A549 and A549/DDP cells labeled with DiR uorescence were injected into the yolk sac by microinjection technology, after 3 days treatment of 5μM OP-B, the uorescence intensity of zebra sh in each group was measured by uorescent stereo-microscope (Fig.2e). Statistics showed that the optical density of the A549 and A549/DDP administration groups was signi cantly lower than that of the model groups (Fig.2f). Similar to the results in mice, proliferation rate of the xenografted tumor in A549/DDP groups was signi cantly lower than that in A549 groups (Fig.1g).
Taken together, OP-B has more signi cant inhibitory effect on DDP-resistant A549 tumors in vivo and in vitro.

OP-B caused signi cant pyroptosis in A549/DDP cells
In order to further explore the reason why A549/DDP cells are more sensitive to OP-B than A549 cells, we took the lung tissues from the mice mentioned in Fig. 3a for transmission electron microscope (TEM) observation. We found that different degrees of swelling, accompanied by the rupture of the cell plasma membrane and the formation of bubble-like protrusions occurred in A549 and A549/DDP cells that in ltrated in lung tissue (Fig. 3a). We speculated that the reason why OP-B signi cantly inhibited the growth of A549/DDP cells compared with A549 cells, which may due to more signi cant pyroptosis of A549/DDP cells.
Next, we used JC-1 mitochondrial membrane potential (MMP) probe to label the above cells. After 24 hours treatment with OP-B (5μM), ow cytometry was used to detect the changes in the number of red and green uorescent cells to quantify the MMP. The results showed that the level of MMP in A549 and A549/DDP cells decreased after OP-B treatment, while it decreased more signi cantly in A549/DDP cells (Fig. 3b, c and d).
Since the occurrence of pyroptosis is accompanied by the release of a large amount of in ammatory substances and decrease of mitochondrial membrane potential, we then tested LDH release rate and the changes of mitochondrial membrane potential (MMP) in A549 and A549/DDP cells. The results showed that after OP-B (5μM and 10μM) treatment for 6, 12 and 24 hours, the release of LDH in both cell lines increased in dose-dependent manner, and it reached peak at 12h (Fig. 3e and f). Among them, the LDH release of A549/DDP cells was signi cantly higher than that of A549 cells.

OP-B induced obvious pyroptosis in A549/DDP cancer in vitro and in vivo
Since the occurrence of pyroptosis is often accompanied by the release of in ammatory factors, we detected the levels of Cox2 and IL-1β in the cells. After OP-B treatment (5μM and 10μM) for 24h, the immuno uorescence results showed that the expression levels of Cox2 and IL-1β in both of the cell lines were signi cantly increased, and the expression level of the two proteins was higher at 10μM of OP-B than that at 5μM (Fig.4a and c). From the statistical results, it was obvious that the expression level of Cox2 and IL-1β in A549/DDP cells was higher than that in A549 cells (Fig. 4b and d). Furthermore, the results of the above were also proved in vivo (Fig. 4e~i).

OP-B induced pyroptosis of A549/DDP cells by activating Caspase-1/GSDMD pathway
In order to further explore the mechanism of OP-B-induced pyroptosis in A549/DDP cells, we detected the related mRNA expression levels of the classic pyroptosis pathway in A549 and A549/DDP cells. After different concentrations of OP-B acted on the two cells for 24 hours, qRT-PCR results showed that the expression level of pyroptosis-related mRNA increased in the both of cells. We found that 2.5μM of OP-B signi cantly upregulated pyroptosis-related mRNA only in A549/DDP cells, while in A549 cells, the concentration of OP-B needs to reach 5μM that can be just to upregulate mRNA. It suggested that A549/DDP cells were more sensitive to OP-B than A549 cells (Fig. 5a). Under the treatment of different concentration, the expression level of pyroptosis-related mRNA in A549/DDP cells was higher than that in A549 cells. It further showed that A549/DDP cells had a higher degree of pyroptosis than A549 cells (Fig.   5b).
In addition, we also tested the expression of related proteins in the classical pathway of pyroptosis under the action of OP-B (Fig. 5c). And to investigate whether Caspase-1 play a vital role in the pyroptosis of A549/DDP cells, the expression of related proteins were detected after adding Caspase-1 inhibitor VX765 (Fig. 5d). We found that under the action of 10μM OP-B, the expression of pyroptosis-related proteins in A549 and A549/DDP cells was signi cantly up-regulated (Fig. 5e), while pyroptosis-related proteins in A549/DDP cells were up-regulated more signi cantly than that in A549 cells (Fig. 5f). After adding the inhibitor VX765, the downstream protein GSDMD-N of the pyroptosis pathway still expressed under the action of OP-B in A549 cells, while in A549/DDP cells it was inhibited (Fig. 5g), which suggested that in A549/DDP cells, the classic pyroptosis pathway of Caspase-1/GSDMD is the main pyroptosis mechanism.
Finally, we detected the expression of the key protein GSDMD-N in A549 and A549/DDP lung tissue of tumor in situ. The immunocytochemistry results further con rmed that OP-B could induce more obvious pyroptosis in A549/DDP tumor in situ ( Fig. 5h and i).

Discussion
Currently, the clinically used comprehensive treatment mainly based on chemotherapy, while the e cacy is not satisfactory. The main reason of it is the emergence of tumor multidrug resistance (MDR) (17). Therefore, it is very urgent to investigate the mechanism of MDR in lung cancer and nd out the effective reversal strategy to solve it. Traditional Chinese Medicine has been paid more and more attention in MDR research of lung cancer due to the advantages of high e ciency, low toxicity and multi-targets.
Radix Ophiopogonis is commonly used in traditional Chinese medicine to treat lung diseases. In various chemical compositions of ophiopogonis, saponin is the key to quality control of it. OP-B, as a saponin compound, had been proved to have obvious anti-lung cancer effect in vivo and in vitro (13)(14)(15). However, whether it has the function of reversing drug resistance of lung cancer cells remains unknown. Herein, through a large number of cell and animal (mice and zebra sh) experiments, we veri ed that OP-B had a signi cant inhibitory effect on DDP resistant A549 cells. Meanwhile, in combination with DDP, OP-B enhanced the sensitivity of A549/DDP cells to DDP.
Further observation of the tumor tissues under electron microscope found that the morphology of the tumor cells in A549/DDP orthotopic tumor models showed more signi cant feature of pyroptotic cell death as bubbles swelling from the plasma membrane and causing lysis and massive release of cellular contents. As occurrence of pyroptosis is always followed by releasing of many proin ammatory factors, including IL-1β, IL-18, ATP, and HMGB1 (18)(19)(20) and excessive Caspase activation (19). Next, detection of pyroptosis related indexes, such as LDH, MMP, Cox2, IL-1β in vivo and in vitro veri ed the hypothesis of pyroptosis, and further investigation of Caspase-1/GSDMD pathway by qRT-PCR and western blot both proved that OP-B induced more signi cant pyroptosis in A549/DDP cells than that in A549 cells.
As tumor cells always show innate resistance to apoptosis, the development of new strategies to induce pyroptosis may provide more e cient cancer therapy options and improve patient survival (21,22). While, there is a lack of research on the relationship between pyroptosis and tumor resistance currently. BI2536 is an inhibitor of PLK1, Wu M et al. combined BI2536 with cisplatin (DDP) to treat ESCC cells and found the combination of two drugs have a synergistic effect on tumor inhibition and can make pyroptotic death of cancer cells by Caspase-3/GSDME pathway. Their further research revealed that BI2536 can reverse DDP resistance of ESCC by inhibiting DNA damage repair and inducing pyroptosis (12).
In our study, the mechanism of the different effect caused by OP-B between A549 and A549/DDP cells was preliminary explored. And the relationship between pyroptosis and drug-resistance in A549/DDP cells still needed to be further investigated in the future.

Conclusions
Our study demonstrated that OP-B suppressed A549/DDP cells growth more signi cantly than A549 cells.
The mechanism of it may involve that OP-B induced A549/DDP cells produced more signi cantly pyroptosis than A549 cells by Caspase-1/GSDMD pyroptosis pathway (Fig. 6). Our nding is helpful to provide new insights into the mechanism of drug-resistant cell death and provide new ideas and directions for the clinical treatment of drug-resistant tumors.  and A549/DDP xenograft after treated with OP-B for 25 days. The bars and error bars indicate the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001 OP-B also had a signi cant inhibitory effect on A549/DDP carcinoma in situ in mice and transplanted tumors in zebra sh. a. Bioluminescent imaging and quanti cation of photon ux of 3mg/kg OP-B treated groups with right lung parenchyma injection of luciferase-marked A549 and A549/DDP cells. b. Before and after OP-B treatment, the uorescence intensity changes on model and OP-B groups of A549 and A549/DDP. c. Tumor growth rate of A549 and A549/DDP calculated according to the uorescence intensity data. d. Comparison of tumor growth rates between A549 and A549/DDP. e. Representative images taken by uorescence stereomicroscope of the zebra sh that were injected with A549 and A549/DDP cells and treated with 5μM OP-B for 3 days. f. Relative uorescence intensity of A549 and A549/DDP tumor in zebra sh after OP-B treatment. g. Comparison of tumor growth rate between A549 and A549/DDP that calculated according to the uorescence intensity data. The bars and error bars indicate the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001 Figure 3 OP-B caused signi cant pyroptosis in A549/DDP cells. a. Transmission electron microscope images of tissue of tumor in situ (A549 and A549/DDP) after 3mg/kg OP-B treatment for 14 days (Yellow triangles represent tumor cells). b~d. Mitochondrial membrane potential of A549 and A549/DDP cells was detected by ow cytometry after 5μM OP-B treatment for 24h. e~f. The level of LDH release in A549 and A549/DDP cells treated with different concentrations of OP-B after 6h,12h and 24h.The bars and error bars indicate the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001  The bars and error bars indicate the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001 Figure 6 Schematic diagram of mechanism of this research. OP-B induced A549/DDP cells pyroptosis by Caspase-1/GSDMD signal pathway.