Abstract
Background/Aim: Cisplatin resistance often leads to treatment futility and elevated mortality rates in patients with lung cancer. One promising strategy to address this challenge involves the integration of traditional Chinese medicine (TCM) with chemotherapeutic drugs. Currently, the potential synergistic effect and underlying mechanism of polyphyllin II (PPII) and cisplatin combination in combating cisplatin (DDP) resistance in lung cancer remain unexplored. Materials and Methods: In this study, we established a cisplatin resistance model using A549 cells and explored the underlying mechanisms of PPII in combination with cisplatin in A549/DDP resistant cells. Specifically, we assessed the impact of PPII combined with cisplatin on A549/DDP cell proliferation, viability, and the expression of apoptosis-related proteins. To gain deeper insights into the underlying mechanism, we examined the effects of PPII and cisplatin on mitochondrial function in A549/DDP cells. Results: This combination induced cell cycle arrest at both the S phase and G2/M phase in A549/DDP cells, thereby promoting apoptosis. Western blotting confirmed that DDP acted synergistically with PPII to enhance the expression of apoptotic proteins, diminish the expression of anti-apoptotic proteins, and promote the expression of anti-proliferation proteins in the mitochondrial pathway of A549/DDP cells. Conclusion: The combination of PPII and cisplatin effectively modulated the mitochondrial function, thereby reversing drug resistance in A549/DDP cells. This innovative combination therapy shows significant promise as a novel strategy for overcoming cisplatin resistance in lung cancer.
Lung cancer stands as the most prevalent and lethal malignancy globally (1). It comprises two major subtypes: small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC), with the latter constituting approximately 80% of cases (2, 3). In recent years, chemotherapy has remained the cornerstone of lung cancer treatment, serving as the primary therapeutic modality employed in clinical practice (4, 5). Unfortunately, a significant proportion of patients with lung cancer eventually develop multidrug resistance (MDR) during their treatment journey. Notably, NSCLC exhibits a lower 5-year survival rate (20-30%) compared to other malignancies (6). These findings underscore the substantial challenges posed by chemoresistance, the primary impediment to effective lung cancer treatment. Addressing chemoresistance is a formidable challenge, necessitating the exploration of potential countermeasures to mitigate its adverse impact on treatment outcomes.
Chemoresistance has persistently plagued the clinical management of lung cancer. The emergence of drug transporter alterations (7-9), altered-drug-metabolizing-enzymes (10-12), epithelial-mesenchymal transition (EMT) (13-16), microenvironmental factors (17, 18), cell cycle arrest and apoptosis (19), as well as autophagy (20), have all been identified as critical drivers of chemoresistance in lung cancer, supported by a growing body of scientific literature.
Paris polyphylla, a medicinal herb renowned for its ability to alleviate heat and detoxify the body, contains various chemical compounds, including steroidal saponins, C21 steroids, flavonoids, polysaccharides, and amino acids (21). Among these constituents, steroidal saponins stand out as the main active components with anti-tumor properties (22). Specifically, polyphyllin II (PPII), a steroidal saponin derived from Paris polyphylla (23), has demonstrated efficacy against various cancer types, including colorectal (24), bladder (25), glioma (26), liver (27), and lung cancers (24, 28), with significant anti-tumor effects (29). While previous studies have highlighted PPII’s potential to reverse resistance to gefitinib, a targeted agent for NSCLC (29), its role in chemotherapy resistance remains underexplored. Therefore, this study aimed to investigate PPII’s potential in overcoming drug resistance in A549/DDP cells and elucidate its molecular mechanisms.
Materials and Methods
Cell lines and cell culture. A549 and A549/DDP cells were procured from Guangzhou Geniou Biotechnology Co., Ltd (Guangzhou, PR China) and maintained in DMEM media supplemented with 10% fetal bovine serum. These cells were cultured in a cell incubator at 37°C with 5% CO2 and maximum humidity, with regular passaging. The final concentration of DDP in A549/DDP cells was 2.1:0.002 mg/ml.
Chemicals and drug preparations. PPII, with a purity of approximately 98%, was purchased from Nantong Feiyu Biotechnology Co., Ltd. (Jiangsu, PR China), whereas cisplatin (DDP) was provided by Hunan Provincial Hospital of Combination of Chinese and Western Medicine (Hunan, PR China). PPII’s chemical structure is shown in Figure 1A. To prepare stock solutions, both chemicals were dissolved in dimethyl sulfoxide (PPII, 500 mg/ml; DDP, 1 mg/ml) and stored at −80°C.
Cisplatin (DDP) was cytotoxic to A549 and A549/DDP cells; polyphyllin II (PPII) and DDP exert a synergistic effect on A549/DDP cells. (A) Chemical structure of PPII. (B) The cytotoxic effect of DDP on parental A549 and resistant A549/DDP was examined using CCK8 assay. (C-H) The cytotoxic effect of PPII and PPII in combination with DDP on A549/DDP cells was assayed using the CCK8 assay. All data are presented as mean±SD for individual experiments performed in triplicate (****p<0.0001; *p<0.05).
CCK8 assays. Resistance index of model. A density of 5×103 A549/DDP and 5×103 A549 cells were seeded into individual wells of a 96-well microplate and incubated for 24 h. The cells were then exposed to various concentrations of DDP (0 μg/ml, 2 μg/ml, 4 μg/ml, 8 μg/ml, 16 μg/ml, 32 μg/ml, and 64 μg/ml) for an additional 24 h in a humidified environment with 5% CO2 at 37°C. Subsequently, each well received 10 μl of CCK-8 reagent (Guangzhou, PR China), followed by 1 h incubation at 37°C in a humidified incubator with 5% CO2, and OD values were recorded at 450 nm using a microplate reader from a multi-mode microplate reader (TECAN, Männedorf, Switzerland). Cell survival (%) was determined by dividing the absorbance values of cells in the treatment group by those in the control group. The IC50 values of DDP were determined using GraphPad Prism version 9.0 software (San Diego, CA, USA), and the resistance index (RI) of A549/DDP was calculated as [RI=IC50 (resistant cells)/IC50 (parental cells)].
Screening of PPII concentrations. A549/DDP cells (5×103 cells/well) were seeded into six 96-well microplates and incubated for 24 h. DDP (IC50 of DDP in A549 cells) and varying concentrations of PPII were added, followed by incubation in a humidified incubator with 5% CO2 at 37°C for 24, 48, and 72 h. CCK-8 reagent was added to each well, and after 1-h incubation, OD values were measured to determine the IC50 of PII in the presence or absence of DDP.
Evaluation of cell viability in response to PPII treatment. A total of 5×103 A549/DDP cells were seeded in a 96-well microplate and incubated for 24-h. Various PPII concentrations (40 μg/ml, 60 μg/ml, and 80 μg/ml) were administered, with the DDP group as the reference control. After 24, 48, and 72 h of incubation, CCK-8 reagent was added, followed by a 1 h incubation at 37°C with 5% CO2. Cell viability was assessed by comparing the absorbance values of the treatment group to the blank group, yielding cell survival rates as percentages.
5-ethynyl-2′-deoxyuridine (EDU) assay. Cell proliferation was assessed using the EDU (5-ethynyl-2′-deoxyuridine) test. A549/DDP cells were incubated with the EDU reagent in the dark for 30 min following fixation and permeabilization. Cell proliferation was visualized and recorded under a fluorescence microscope (ZEISS, Oberkochen, Germany).
Colony formation assay. The DDP-resistant lung adenocarcinoma cell line A549/DDP was seeded at a density of 1,000 cells/well in a 6-well plate. After 24 h of incubation, the medium was replaced with the drug-containing medium. After two weeks, the medium was removed, and the cells were washed twice with phosphate-buffered saline (PBS). The cells were fixed with 4% paraformaldehyde at 25°C for 30 min, washed with PBS, and stained with crystal violet for 10-20 min. After additional PBS washes and air drying, photographs of the entire 6-well plate or individual colonies were obtained.
Flow cytometry analysis for apoptosis. DDP-resistant A549/DDP cells were seeded in a six-well culture plate (2.5×105 cells per well) and incubated for 24 h. Various PPII doses (0, 40, 60, and 80 μg) were added in combination with DDP for 24 h. Following treatment, the cells were harvested, suspended in binding buffer, and double stained with 5 μl Annexin-V FITC and 5 μl propidium iodide (PI) (100 μg/ml) for 20 min at room temperature in the dark. Stained cells were resuspended in 500 μl of binding buffer and analyzed using a flow cytometer (Beckman, Brea, CA, USA). Data analysis was performed using FlowJo software (Stanford University, Stanford, CA, USA).
Cell cycle analysis. A549/DDP cells were initially seeded in 6-well culture plates at a density of 2.5×105 cells per well and allowed to incubate for 24 h. Subsequently, the cells were exposed to varying doses of PPII (0, 40, 60, and 80 μg) for 24 h. After treatment, the cells were washed with PBS and fixed in 70% ethanol for 30 min at 4°C. Following this step, the ethanol was removed by centrifugation, and the cells were rinsed twice with PBS. The resulting cell pellet was then incubated with 300 μl of 1×PI staining solution for 30 min at 37°C in the dark. The PI fluorescence was excited using a 488 nm laser using a Backman flow cytometer to analyze cell cycle distribution, including phases, such as sub-G1, G1, S, and G2/M.
Tunel apoptosis test. Apoptosis was detected using the TUNEL Apoptosis Detection Kit (Boster, Wuhan, PR China). A549/DDP cell slides were prepared in six-well plates with 2.5×105 cells per well and treated with medication for 24 h once the slide surfaces were stabilized. Adherent cells were fixed with 4% paraformaldehyde and exposed to 3% H2O2 for 10 min. Following this step, each group received the addition of 20 μl of the TUNEL Reaction Solution, which was composed of 1 μl of TdT, 1 μl of dUTP, and 18 μl of labeling buffer., in a humid environment for 2 h. Next, the cells were incubated at 37°C for 30 min with diluted Streptavidin-Biotin Complex (SABC), stained with Diaminobenzidine (DAB), mildly counterstained with Hematoxylin, and washed, dried, and mounted on slides. Finally, samples were examined under a microscope and captured using a camera.
Electron microscopy. After a 24 h incubation, individual cells from each group were collected and washed with 0.1 M phosphate buffer (pH 7.4). Subsequently, the cells were fixed at 4°C for 2 h in an electron microscope osmium tetroxide, after which the samples were postfixed in 1% aqueous osmium tetroxide. After washing with PBS, cells underwent gradual dehydration in a series of graded ethanol solutions (30% to 100%), Encased within an epoxy matrix, the samples were subjected to a controlled incubation at 60°C for a duration of 48 h. Following this, semithin sections measuring 1.0 mm in thickness were rendered visible through the application of toluidine blue staining. Subsequently, ultrathin sections of the cell samples were stained with lead citrate and uranyl acetate. Finally, the intricate ultrastructural images were meticulously captured using a high-resolution transmission electron microscope (Hitachi, Tokyo, Japan).
Jc-1 probe fluorescence detection. A549/DDP cells were cultivated in six-well plates and subjected to drug treatments. After treatment, cells were washed twice with PBS and were subsequently treated with JC-1 dye for 20 min. Following this incubation, the cells were washed with PBS and placed in phenol red-free media for fluorescence microscopy analysis. The fluorescence emitted by JC-1 dye was observed under a microscope, and images were captured to assess changes in mitochondrial membrane potential.
Glucose content test. For the glucose assay, A549/DDP cells were first cultured in six-well plates until they adhered. Subsequently, the cells underwent a 24-h drug treatment. An intracellular glucose assay kit (Jiancheng Bioengineering Co., Ltd., Nanjing, PR China) was used to quantify the glucose content. In brief, the cells were mechanically disrupted in 50 μl of lysis buffer, and 2.5 μl of each sample was added to individual wells, along with 252.5 μl of the test working solution (250 μl). The plate was then incubated at 37°C for 10 min. Finally, the absorbance at 505 nm wavelength was measured using a multi-mode microplate reader (ZEISS, Oberkochen, Germany).
ATP content test. For the ATP detection assay, cells were seeded in six-well plates at a density of 2.0×105 cells/well. Once the cells reached 60% confluence, they were treated with the drugs for 24 h. To measure intracellular ATP levels, an ATP test kit (Box Sangon Technology Co., Ltd., Beijing, PR China) was used. ATP extracts were obtained by mechanically disrupting the cells, and cell supernatants were collected through centrifugation at 10,000×g and 4°C. In a 96-well culture plate, the prepared ATP detection working solution and the extracted ATP samples were simultaneously added in a 96-well culture plate, and thoroughly mixed. Absorbance values at 340 nm were measured at two-time points: 10 s (recorded as A1) and 180 s (recorded as A2). ATP levels in cells were then calculated using the ATP content equation (Box Biotech, Beijing, PR China).
Western blot. After a 24-h drug treatment, total protein was extracted from A549/DDP cells using RIPA lysis buffer (Kangwei Biotech Co., Beijing, PR China), and protein concentration was determined using a bicinchoninic acid assay kit (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer’s instructions. Equal amounts of protein were resolved on a 12% SDS-PAGE gel and subsequently transferred to PVDF membranes. Membranes were blocked with TBST buffer (20 mmol/l Tris-HCl, 150 mmol/l NaCl, 0.05% Tween 20) containing 5% skimmed milk for 1 h at room temperature. Subsequently, membranes were incubated overnight at 4°C with gentl shaking with primary antibodies against caspase-3, Bax, Bcl-2, AMPK, mTOR, β-actin, and PPAR provided by Sanying Biotechnology Co., Ltd. (Sanying Biotech, Wuhan, PR China). After three washes with TBST buffer, membranes were incubated with anti-mouse IgG-HRP and anti-rabbit IgG-HRP secondary antibodies for 2 h. Target proteins were visualized using a commercial ECL kit (Kangwei Biotech Co., Beijing, PR China), and protein bands were captured with an imaging system (General Electric, Boston, MA, USA). Band intensities were quantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA), and the relative expression levels of target proteins were normalized to the loading control actin. Statistical analysis was performed to compare differences in protein expression levels between the treatment and control groups.
Statistical analysis. Statistical significance was determined using Graphpad Prism 9.0 (CA, USA) analysis software. Results are presented as the mean±standard deviation (SD) of three independent experiments. A value of p<0.05 was considered to indicate statistical significance., indicating that the observed differences between groups are unlikely to have occurred by chance. Consent for publication was obtained from the participant.
Results
Establishment of resistant cell models. The drug resistance model was established by treating parental A549 cells and resistant A549/DDP cells with varying concentrations of DDP and assessing the drug resistance index of A549/DDP cells. The results revealed that the IC50 in the parental A549 cells was 9.917 μg/ml, whereas that of resistant A549/DDP cells was 49.39 μg/ml. This indicated that the drug resistance index of A549/DDP cells was approximately 5, classifying them as moderately resistant cells. These findings confirm the successful establishment of the drug resistance model (Figure 1B).
PPII combined with DDP inhibits the growth of A549/DDP cells. To assess the PPII’s potential in reversing drug resistance in A549/DDP cells, we examined its toxicity on resistant cells, both alone and in combination with DDP (Figure 1C-E). PPII alone exhibited a dose-dependent cytotoxic effect on A549/DDP cells, with IC50 values of 400 μg/ml at 24 h, 376.2 μg/ml at 48 h, and 337 μg/ml at 72 h. When combined with a low dose of cisplatin (IC50 concentration of cisplatin from parental A549 cells, 9.917 μg/ml), the IC50 values for the combination treatment on A549/DDP cells at 24 h, 48 h, and 72 h significantly decreased to 61.22 μg/ml, 50.98 μg/ml, and 40.53 μg/ml, respectively. These results suggest that PPII effectively enhances the sensitivity of resistant A549/DDP cells to DDP. This approach, involving a constant DDP IC50 concentration, ensures that any changes in cell viability are primarily due to the effects of PPII, rather than changes in DDP concentration. Selecting the PPII IC50 concentration from the PPII synergistic DDP proliferation assay at 24 h is a logical choice, as it represents a concentration at which PPII significantly affects cell viability when combined with DDP. Overall, our findings indicate that PPII sensitizes A549/DDP cells to DDP in a dose- and time-dependent manner. At the constant DDP concentration of 49.39 μg/ml, the survival rate of A549/DDP cells remained unchanged over time. However, when PPII was added, the cell survival rate decreased in a time- and dose-dependent manner, suggesting that PPII may effectively reverse the resistance of A549/DDP cells to DDP. These promising results imply that PPII holds potential as an adjuvant therapy for the treatment of DDP-resistant NSCLC. Furthermore, we evaluated the effect of PPII in combination with DDP on A549/DDP cell proliferation using EDU proliferation and colony formation assays. Our data consistently showed that PPII dose-dependently inhibited A549/DDP cell proliferation and colony formation, providing robust support for the potential of PPII to restore sensitivity to DDP in DDP-resistant A549/DDP cells. To further investigate the impact of PPII in combination with DDP on A549/DDP cell proliferation, we conducted EDU proliferation and colony formation assays (Figure 2A and B). The results consistently demonstrated that PPII effectively suppressed the proliferation and colony formation of A549/DDP cells in a dose-dependent manner. These findings provide compelling evidence that PPII can restore sensitivity of DDP-resistant A549/DDP cells to DDP.
Polyphillin II (PPII) synergizes with cisplatin (DDP) to inhibit proliferation of A549/DDP cells. (A) The effect of DDP and DDP in combination with PPII was examined using the EDU assay. (B) The results of colony formation assay after treatment with DDR or PPII in combination with DDP. (C) Statistical analysis of the results of the EDU proliferation assay. (D) Statistical analysis of results of the colony formation experiments. All data are presented as mean±SD for individual experiments performed in triplicate (****p<0.0001; *p<0.05).
PPII induced cell cycle arrest and apoptosis in A549/DDP cells. To elucidate the mechanism of the anticancer effects of the combination of PPII and DDP (PPII+DDP), we investigated the impact of this on the distribution of cells in the various phases of the cell cycle and apoptosis (Figure 3B). Notably, there was a substantial increase in the proportion of cells in the S and G2/M phases after the administration of PPII in combination with DDP. These findings strongly suggest that the treatment with PPII induced a cell cycle arrest at the S and G2/M phases in A549/DDP cells. Additionally, there was a significant elevation in the proportion of cells in the sub-G1 phase, suggesting that the combination of PPII and DDP has pro-apoptotic effects. To further substantiate the relationship between PPII and apoptosis, we assessed the extent of PPII-induced apoptosis. As illustrated in Figure 3A, the percentage of apoptotic cells exhibited a remarkable increase with escalating doses of PPII treatment. In the control group, the apoptotic cell population was merely 3.09%, whereas following DDP treatment the percentage of apoptotic cells reached 6.38%. Remarkably, the proportion of apoptotic cells substantially surged to 10.46%, 18.96%, and 26.1% following treatment with the combination of DDP and PPII at concentrations of 40 μg, 60 μg, and 80 μg, respectively. Furthermore, the TUNEL apoptosis assay confirmed the pro-apoptotic effect of PPII in combination with DDP on A549/DDP cells (Figure 3C). Additionally, Western-blot analysis revealed a significant upregulation in the expression of caspase-3 and Bax proteins, accompanied by a downregulation in the expression of the bcl-2 protein upon treatment with the combination of PPII and DDP. These findings strongly suggest that PPII enhances the apoptotic effect of DDP in A549/DDP cells by modulating the caspase-3/Bax/bcl-2 signaling pathway.
Polyphillin II (PPII) synergizes with cisplatin (DDP) to induce apoptosis. (A-B) Apoptosis and cell cycle were detected using flow cytometry after treatment with the combination of PPII with DDP. (C) Apoptosis was further verified after PPII synergized with DDP treatment. (E) Western-blot validation of relevant apoptotic protein levels. (F) Statistical analysis of the apoptosis results obtained using flow cytometry. (H) Statistical analysis of the western-blot results. (G) Statistical analysis of cell cycle results obtained using flow cytometry. (I) Statistical analysis of the apoptosis results obtained using the TUNEL apoptosis assay. The results are presented as mean±SD of three independent experiments, *p<0.05, ****p<0.0001.
PPII induces apoptosis through mitochondrial dysfunction. To elucidate the underlying anticancer mechanism of PPII, we investigated its impact on mitochondrial function in A549/DDP cells when applied in combination with DDP. We conducted a transmission electron microscopy (TEM) analysis to assess mitochondrial morphology. In the control group, mitochondria displayed predominantly intact morphology with well-organized cristae. However, when treated with DDP alone, mitochondrial morphology remained relatively intact, with minor alterations. Remarkably, with increasing concentrations of PPII, we observed progressive exacerbation of mitochondrial damage was observed. The mitochondria appeared pyknotic with disorganized or even absent cristae, along with signs of mitochondrial membrane swelling (Figure 4A). Furthermore, we assessed alterations in mitochondrial membrane potential (MMP) using JC-1 staining. A decrease in MMP serves as an indicator of early apoptosis and mitochondrial dysfunction. Our findings demonstrated that the combination of PPII and DDP led to a substantial induction of MMP loss, signifying impaired mitochondrial function (Figure 4B). Moreover, we measured cellular levels of ATP and glucose as indicators of mitochondrial function and energy metabolism (Figure 4D and E). Notably, following treatment with the combination of PPII and DDP, glucose and ATP levels significantly decreased, indicating impaired mitochondrial energy production. Based on the obtained results, it can be concluded that the combined treatment with the combined of PPII with DDP has the potential to re-sensitize A549/DDP cells to DDP by inducing mitochondrial damage, which leads to abnormal energy metabolism. This conclusion is supported by the observed changes in mitochondrial morphology, loss of mitochondrial membrane potential, and decreased levels of ATP and glucose content. Furthermore, our findings suggest that PPII treatment may disrupt the mitochondrial electron transport chain, leading to decreased ATP production, cellular energy depletion, and ultimately, apoptosis. Further studies are warranted to explore the underlying molecular mechanisms of PPII-mediated mitochondrial dysfunction and to determine its therapeutic potential in DDP-resistant lung cancer.
Polyphillin II (PPII) synergizes with cisplatin (DDP) to activate the AMPK pathway, affect mitochondrial energy metabolism, and induce apoptosis. (A) Mitochondrial morphological changes observed using electron microscope. (B) Mitochondrial membrane potential changes using JC-1 staining. (C) Western blot results. (D) Glucose content results. (E) ATP content changes. (F) Statistical analysis of the results obtained using JC-1 as a mitochondrial membrane potential indicator. (G-H) Statistical analysis of the western-blot results. Results are presented as mean±SD of three independent experiments, *p<0.05, ****p<0.0001.
Involvement of the AMPK-mTOR signaling pathway in PPII and DDP-induced apoptosis. AMPK, recognized as an energy sensor, plays a crucial role in maintaining energy homeostasis (30). Given that mitochondria serve as the primary organelles responsible for energy production, any mitochondrial damage can lead to reduced levels of ATP (31) and glucose (32), consequently activating AMPK (33). To investigate the impact of PPII on the AMPK signaling pathway, we analyzed the phosphorylation status of AMPK and its downstream signaling pathways, with particular emphasis on the molecule mTOR, a major regulator of the AMPK pathway (34). Western blot analysis demonstrated that PPII treatment induced a significant increase in the phosphorylation of AMPK, subsequently inhibiting the phosphorylation of mTOR, an integral downstream effector of the AMPK signaling pathway (34). This activation of the AMPK signaling pathway resulted in the activation of its downstream anti-proliferation protein, PPARγ (Figure 4C).
Discussion
Despite the exploration of numerous platinum-based compounds, DDP chemotherapy remains the gold standard for treating non-small cell lung cancer (35, 36). However, the emergence of chemoresistance poses a significant challenge, substantially limiting DDP’s effectiveness in non-small cell lung cancer patients (37-40). Overcoming this chemoresistance is vital to improve patient outcomes and survival. Therefore, efforts to discover effective strategies for overcoming chemoresistance in patients with lung cancer hold significant clinical relevance. The exploration of the anticancer mechanisms of active compounds derived from traditional Chinese medicine has garnered increasing attention in the medical field. Previous studies have highlighted various compounds, such as Asiatic acid (41), Shenmai injection (42), and ginsenoside RG3 (43), for their potential to reverse DDP resistance in A549/DDP cells through diverse molecular mechanisms. The extensive clinical utilization of traditional Chinese medicine offers a promising avenue for anticancer therapy in patients with limited response to chemotherapy. Natural products, including those derived from traditional Chinese medicine, are increasingly recognized as valuable reservoirs for discovering novel therapeutic agents in drug development. In the present study, we demonstrated that PPII effectively re-sensitizes DDP-resistant NSCLC cells to DDP, exhibiting significant efficacy with reduced toxicity in vivo. Recent studies have highlighted the relationship between chemoresistant NSCLC cells and high mitochondrial bioenergetics, which is associated with shifts in metabolic pathways (44, 45). Considerable evidence suggests that a decrease in ATP content, reduced glucose content, and disruption of mitochondrial membrane integrity play an important role in programmed cell death (46, 47). AMPK functions as an energy sensor (48) and plays a crucial role in mitochondrial biogenesis in NSCLC (49, 50). AMPK activation is a response to energy depletion and regulates and regulates the metabolism of proteins, (51), lipids (52), carbohydrates (53), as well as autophagy (54), and mitochondrial homeostasis (39). It encompasses nearly the entirety of physiological metabolic activity in living organisms (55). AMPK activation can prevent tumor cell proliferation and promote apoptosis (56) AMPK and the mammalian target of rapamycin (mTOR) are two evolutionarily conserved kinases that collectively regulate almost every aspect of cellular and systemic metabolism (57). AMPK examines mTOR activity and increasing glucose energy production through glycolysis and mitochondrial oxidative phosphorylation, acting as a catabolic enzyme (58). The role of peroxisome proliferator-activated receptor gamma (PPARγ), as a regulator of adipocyte differentiation, glucose metabolism, and lipid homeostasis, is well-established (59). In tumor systems, PPARγ impedes tumor development and progression by regulating cancer cell differentiation, proliferation, and apoptosis through various molecular pathways (60-64). This study revealed a synergistic effect of PPII in combination with DDP on A549/DDP cells, leading to significant activation of the AMPK signaling pathway, coupled with mTOR inhibition. This molecular interplay facilitated the upregulation of PPARγ, a downstream protein, ultimately leading to the suppression of cell proliferation and the induction of apoptosis. Furthermore, the combined treatment with PPII and DDP exhibited inhibitory effects on oxidative phosphorylation, causing a decline in mitochondrial membrane potential. These findings strongly suggest that the apoptotic response triggered by the combination of PPII in combination with DDP is closely associated with mitochondrial impairment. Existing literature substantiates the notion that therapeutic agents modulate key cellular processes, including proliferation, apoptosis, autophagy, and metabolism, through the mediation of the AMPK/mTOR pathway (63, 65-67). Our observations indicate that PPII synergizes with DDP, enhancing its antiproliferative and proapoptotic effects in A549/DDP cells. This synergism is attributed to the activation of AMPK signaling and the concomitant inhibition of mTOR phosphorylation, ultimately upregulating PPARγ and suppressing cell growth. Furthermore, our findings suggest that PPII and DDP collaborate to disrupt mitochondrial energy metabolism, resulting in mitochondrial membrane potential loss and the activation of the intrinsic apoptotic pathway via the regulation of Bcl-2 family proteins and caspase-3.
Our results underscore the potential of PPII as an adjunct therapy for DDP-resistant NSCLC, emphasizing the significance of mitochondrial dysfunction in chemoresistance. Targeting mitochondrial function and bioenergetics represents a promising therapeutic approach to overcoming chemoresistance in NSCLC and enhancing the efficacy of existing chemotherapies (Figure 5).
The proposed mechanism of the effect of PPII on A549/DDP cells. PPII promotes apoptosis by damaging mitochondria.
Footnotes
Authors’ Contributions
Conceived and designed study: Lian Peng, Renyi Yang. Collected data: Zhibing Wang, Huiying Jian. Analysis and interpretation of data: Xiaoning Tan, Jian Li, Drafting and critically revising: Zuomei He, Rui Huang.
Funding
This work was supported by National Natural Science Foundation of China (82205227), Natural Foundation of Hunan Provincial (2023JJ30448, 2021JJ40310), Science and Technology Project of Traditional Chinese medicine in Hunan Province (2020173, D2022064), Natural Science Foundation of Changsha (kq2202264), Graduate Hunan University of Traditional Chinese Medicine Innovation (2022CX54, 2022201, 2023CX26).
Conflicts of Interest
The Authors affirm that they possess no discernible conflict of financial interests or personal relationships that may have had the potential to impart bias or exert undue influence on the findings presented within this manuscript.
- Received June 8, 2023.
- Revision received October 22, 2023.
- Accepted October 24, 2023.
- Copyright © 2024 The Author(s). Published by the International Institute of Anticancer Research.
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).
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