Piperlongumine decreases cell proliferation and the expression of cell cycle- associated proteins by inhibiting Akt pathway in human lung cancer cells

Abstract

Piperlongumine (PL), an alkaloid derived from a Southeast Asian pepper plant, is recognized for its selective cytotoxic effects against various cancer cell types. To investigate its potential anti-lung cancer properties, A549 cells were treated with PL at concentrations ranging from 0 to 40 μM for 24 hours.

Following treatment, changes in the expression levels of proteins associated with the cell cycle, including cyclin D1, cyclin-dependent kinase 4 (CDK4), CDK6, and retinoblastoma protein (Rb), were assessed. Additionally, the study examined key intracellular signaling molecules such as extracellular signal-regulated kinase 1/2 (ERK1/2), Akt, p38, and nuclear factor-κB (NF-κB) using Western blot analysis.

The results demonstrated that PL significantly reduced cell proliferation in a dose-dependent manner. Flow cytometric analysis revealed an increased proportion of cells arrested in the G1 phase in the group treated with 40 μM of PL. Furthermore, levels of reactive oxygen species (ROS) were significantly elevated in cells exposed to 20–40 μM PL.

PL treatment at 40 μM led to a marked reduction in the expression of cyclin D1, CDK4, CDK6, and phosphorylated Rb. The phosphorylation of Akt was notably suppressed, while ERK1/2 phosphorylation was enhanced. Additionally, PL significantly reduced the nuclear translocation of NF-κB p65.

These findings suggest that Piperlongumine exhibits antiproliferative effects in A549 lung cancer cells, potentially through modulation of cell cycle regulators, induction of oxidative stress, and alteration of key signaling pathways.

Introduction

Lung cancer remains one of the leading causes of mortality worldwide. Despite advancements in therapeutic strategies such as surgery, chemotherapy, and radiotherapy, the five-year survival rate for lung cancer patients continues to be dismally low, remaining under 15 percent. This poor prognosis, combined with the limited effectiveness and significant side effects of current treatments, has prompted increased attention toward natural products as alternative anticancer agents.

Carcinogenesis involves a range of genetic alterations, with one of the most prominent being abnormal upregulation of cell proliferation, a key factor in tumor development. The process of cell proliferation relies on the timely formation of cyclins and cyclin-dependent kinases (CDKs), which regulate the progression of the cell cycle. Cyclins act as regulatory subunits, binding and activating catalytic partners such as CDK4 and CDK6. The interaction among cyclin D1, CDK4, and CDK6 plays a crucial role in controlling the G1-to-S phase transition, initiating cell cycle progression.

Cyclin D1, in particular, accumulates to its peak level to promote this transition in quiescent cells. Its overexpression has been shown to drive cell cycle progression and enhance tumorigenesis, as observed in rat fibroblasts. In parallel, the retinoblastoma (Rb) protein serves as a key regulator of the cell cycle by controlling the G1 checkpoint and preventing premature entry into the S phase.

Reactive oxygen species (ROS) are also closely linked to cell cycle regulation. While moderate ROS levels can stimulate cell proliferation and differentiation, excessive ROS production leads to oxidative damage and disrupts various cellular signaling pathways. Among these, the mitogen-activated protein kinases (MAPKs) and Akt signaling pathways are critical regulators of cellular responses to stress, influencing both cell proliferation and apoptosis.

Akt, once activated by phosphorylation, promotes cell survival by inhibiting apoptosis and autophagy. It triggers a series of downstream effects, including the activation of anti-apoptotic proteins and prevention of cytochrome c release. Abnormal Akt activation has been implicated in the development and progression of various cancers, including lung, colorectal, and breast cancer, largely by supporting cell cycle progression and suppressing programmed cell death. This makes Akt an attractive molecular target for cancer therapy.

In contrast, activation of the ERK pathway by chemopreventive agents can lead to antiproliferative outcomes, including apoptosis, cellular senescence, and autophagy. The transcription factor nuclear factor kappa B (NF-κB) is another central player in cancer development. It contributes to immune responses, malignant transformation, and cell survival. Upon dissociation from its inhibitor, IκB, NF-κB translocates into the nucleus, where it promotes cancer cell proliferation. This pathway is frequently found to be constitutively activated in various human cancers, including lung cancer.

Previous research has demonstrated that several natural compounds with anticancer properties exert their effects by inhibiting the NF-κB signaling pathway, thereby reducing the proliferation of lung cancer cells.

Piperlongumine (PL), a bioactive alkaloid isolated from the edible pepper plant *Piper longum L.*, exhibits a wide range of biological effects. These include anti-inflammatory, anti-atherosclerotic, antibacterial, and anticancer activities. It has also shown antiplatelet properties due to its antioxidant capacity. Of particular interest is PL’s ability to selectively induce cell death in cancer cells while sparing normal cells. This selective cytotoxicity is largely attributed to increased ROS production and the induction of apoptotic pathways, as shown in various cancer cell lines such as MCF7, HCT116, and A549.

Despite the established anticancer potential of PL, its effects on the expression of cell cycle-regulating proteins and related signaling pathways have not been fully characterized in A549 lung cancer cells. Therefore, the current study aimed to elucidate the cellular mechanisms underlying PL-induced inhibition of cell proliferation in A549 cells. This investigation focused on the modulation of key cell cycle proteins, including cyclin D1, CDK4, CDK6, and phosphorylated Rb, along with the involvement of other critical signaling molecules.

Materials and methods

Materials

RPMI-1640 medium and fetal bovine serum (FBS) were procured from PAN-Biotech (Aidenbach, Germany). Trypsin, phosphate-buffered saline (PBS), and a penicillin/streptomycin solution were obtained from Gibco (Grand Island, NY). Piperlongumine (PL) was purchased from Indofine Chemical Company (Somerville, NJ).

Trypan blue solution and N-acetylcysteine (NAC) were sourced from Amresco (Solon, OH). Dimethyl sulfoxide (DMSO) and 2′,7′-dichlorofluorescin diacetate (DCF-DA) were acquired from Sigma (St. Louis, MO).

Primary antibodies against CDK4 (sc-260), CDK6 (sc-177), cyclin D1 (sc-753), NF-κB p65 (sc-372), phosphorylated Akt (p-Akt, sc-7985-R), glyceraldehyde 3-phosphate dehydrogenase (GAPDH, sc-25778), and lamin B (sc-6216) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Secondary antibodies, including goat anti-rabbit IgG-HRP (sc-2030) and donkey anti-goat IgG-HRP (sc-2020), as well as the chemical inhibitors LY294002 (an Akt inhibitor, sc-201426) and PD98059 (an ERK1/2 inhibitor, sc-3532), were also obtained from the same supplier.

Additionally, antibodies specific to phosphorylated ERK1/2 (p-ERK1/2, #9101), total ERK1/2 (#9102), total Akt (#9272), phosphorylated retinoblastoma protein (p-Rb, #9307), phosphorylated p38 (p-p38, #9215), and total p38 (#9212) were purchased from Cell Signaling Technology (Danvers, MA).

Cell culture and treatments

The A549 cell line, which is derived from human lung adenocarcinoma, was procured from the American Type Culture Collection located in Manassas, Virginia. These cells were cultivated in a specific growth medium known as RPMI-1640. To ensure proper cell growth and maintenance, this medium was further enriched with the addition of 10% fetal bovine serum and a combination of penicillin and streptomycin to prevent contamination. The cells were kept in a controlled environment at a temperature of 37 degrees Celsius within a humidified incubator that contained 5% carbon dioxide.

When the cultured cells reached approximately 50% confluence, indicating a certain level of cell density in the culture vessel, a change in the growth medium was performed. The original medium was replaced with RPMI-1640 medium containing a reduced concentration of fetal bovine serum, specifically 1%. This adjustment in serum concentration was implemented to synchronize the cells, bringing them to a more uniform stage in their cell cycle. Following this overnight incubation in the low-serum medium, the cells were then subjected to treatment with a substance designated as PL, which was dissolved in medium containing the standard 10% fetal bovine serum.

The specific concentrations of PL used in these treatments were carefully chosen based on prior assessments of their impact on cell viability, ensuring that the chosen concentrations were relevant for studying the effects of PL on the cells without causing excessive cell death. In some experimental conditions, the cells underwent a pretreatment step with N-acetylcysteine, commonly referred to as NAC. This pretreatment with NAC was carried out for a duration of one hour before the subsequent application of the PL treatment. Similarly, in other experimental setups, pharmacological inhibitors, such as PD98059 and LY294002, were introduced to the cell cultures for a one-hour period prior to the commencement of the PL treatments.

To illustrate the timing of these treatments, consider an instance where cells were first pretreated with either NAC or one of the pharmacological inhibitors for a duration of one hour. Following this pretreatment period, the PL treatment was then applied to the cells and maintained for a period of 24 hours. In such cases, the total cumulative duration for which the cells were exposed to either NAC or the pharmacological inhibitors extended to 25 hours, encompassing both the initial one-hour pretreatment and the subsequent 24-hour period during which PL was also present. This detailed timeline is crucial for understanding the experimental conditions and interpreting the resulting cellular responses.

Cell proliferation tests

Cell proliferation was determined using trypan blue dye exclusion test. Cells were seeded in 6-well plates, followed by pretreatment of NAC (5 mM, 1 h) and then PL treatments (0–40 μM, 24 h). The live cells were manually counted in the mixture of cells and trypan blue solution using a hemacytometer (Hausser Scientific, Horsham, PA).

Assessment of ROS levels

The generation of reactive oxygen species within the cells was assessed through the utilization of the fluorescent probe 2′,7′-dichlorofluorescein diacetate, commonly known as DCF-DA. This method was conducted following a procedure that has been detailed in prior research publications, specifically in Liu and colleagues’ work from the year 2014. In brief, the A549 cells, which were cultured in six-well plates, underwent an initial one-hour pretreatment with a 5 millimolar concentration of N-acetylcysteine, abbreviated as NAC. Subsequently, these cells were exposed to varying concentrations of the substance PL, ranging from 0 to 40 micromolar, for a duration of one hour.

Following these treatments, the cells were incubated with DCF-DA at a final concentration of 15 micromolar. This was achieved by adding 6 microliters of a 5 millimolar DCF-DA stock solution into 2 milliliters of the cell culture media within each well. The incubation with DCF-DA lasted for 30 minutes. After this incubation period, the cells were carefully washed three times using cold phosphate-buffered saline to remove any unbound DCF-DA. The level of reactive oxygen species produced within the cells was then estimated through observation using an Olympus IX71 fluorescence microscope. Images exhibiting the fluorescence signal were captured digitally using an Olympus DP71 camera and the associated DP controller software, both products of Olympus Optical Co. Ltd., located in Tokyo, Japan.

The cellular areas displaying positive staining for DCF-DA, indicated by green fluorescence, were quantified using Image J software, a tool developed by the National Institutes of Health in Bethesda, Maryland. The resulting quantitative data was then used to generate graphical representations. For a more comprehensive analysis of cellular oxidative stress, cells that had been incubated with DCF-DA at a final concentration of 15 micromolar were also examined for reactive oxygen species production using a CytoFLEX flow cytometer, manufactured by Beckman Coulter in Indianapolis, Indiana. In each experimental treatment group, a total of 25,000 cells were counted. The cells exhibiting positive staining for DCF-DA were identified and further analyzed using the CytExpert software, also a product of Beckman Coulter in Indianapolis, Indiana. This flow cytometric analysis provided a quantitative assessment of the proportion of cells experiencing oxidative stress within each treatment condition.

Cell cycle analysis

The cells were subjected to treatment with the substance PL at varying concentrations, ranging from 0 to 40 micromolar, for a duration of 24 hours. To investigate the potential involvement of oxidative stress in the cellular response to PL, a subset of cells underwent a pretreatment with N-acetylcysteine, or NAC, at a concentration of 5 millimolar for one hour prior to the application of the PL treatments. Following the completion of these treatments, the cells were fixed using a 70% ethanol solution and maintained at a temperature of -20 degrees Celsius for a period of 20 minutes to preserve their cellular structure.

Subsequently, the fixed cells were resuspended in 500 microliters of phosphate-buffered saline. To eliminate any potential interference from RNA, 50 microliters of ribonuclease, resulting in a final concentration of 2 milligrams per milliliter, was added to the cell suspension, and the mixture was incubated for 30 minutes. Following this RNA digestion step, the cells were stained with propidium iodide at a concentration of 50 micrograms per milliliter, yielding a final concentration of 0.1 milligrams per milliliter. This staining procedure was carried out in the dark at room temperature for a duration of 30 minutes, as propidium iodide is a fluorescent dye that binds to DNA and its fluorescence is enhanced upon binding.

After the staining process, the cells were analyzed using a CytoFLEX flow cytometer to determine the distribution of cells across the different phases of the cell cycle. For each experimental sample, the DNA content of at least 25,000 individual cells was measured. The resulting data, reflecting the DNA content of these cells, was then used to calculate the percentage of cells present in each distinct phase of the cell cycle. This analysis was performed using the CytExpert software, a product of Beckman Coulter located in Indianapolis, Indiana, allowing for a quantitative assessment of how the PL treatment, with or without NAC pretreatment, affected the progression of cells through the various stages of the cell division cycle.

Preparation of cell lysate, SDS-PAGE and Western blot analysis

The cultured cells were subjected to a lysis procedure using RIPA buffer, a solution specifically formulated to disrupt cell membranes and release intracellular contents. This buffer contained several components at defined concentrations: 50 millimolar Tris with a pH of 8.0, 150 millimolar sodium chloride, 1% Triton X-100, 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate, commonly known as SDS. To prevent the degradation of proteins released during lysis, a protease inhibitor mixture was included in the buffer, containing 2 micrograms per milliliter of aprotinin, 10 micrograms per milliliter of leupeptin, 1 microgram per milliliter of pepstatin A, 1 millimolar phenylmethylsulfonyl fluoride or PMSF, 5 millimolar ethylenediaminetetraacetic acid or EDTA, 1 millimolar ethylene glycol-bis(β-aminoethyl ether)-N,N,N’,N’-tetraacetic acid or EGTA, 10 millimolar sodium fluoride, and 1 millimolar sodium orthovanadate.

Following the lysis procedure, the resulting cell lysates were centrifuged at a high speed of 21,000 times the force of gravity for a duration of 20 minutes at a temperature of 4 degrees Celsius. This centrifugation step was performed to separate the soluble protein fraction from cellular debris and insoluble components. The supernatant, which contained the solubilized proteins, was carefully collected and transferred to new tubes. The concentration of protein within these supernatant samples was then determined using the Bradford reagent, a commercially available dye-based assay obtained from Sigma-Aldrich, located in St. Louis, Missouri, USA. The protein samples were subsequently stored at a very low temperature of -80 degrees Celsius until further analysis was required.

For protein analysis, specific quantities of the protein samples, precisely 30 micrograms per well, were separated based on their molecular weight using a technique called sodium dodecyl sulfate-polyacrylamide gel electrophoresis, or SDS-PAGE. Following the electrophoretic separation, the proteins were transferred from the polyacrylamide gel onto nitrocellulose membranes. These membranes were then subjected to a blocking step, where they were incubated with a 3% non-fat milk buffer for a period of one hour. This blocking step is crucial to prevent non-specific binding of antibodies in subsequent steps.

After the blocking procedure, the membranes were incubated overnight at a temperature of 4 degrees Celsius with primary antibodies, which are specific to the target proteins of interest. Following several washing steps to remove any unbound primary antibodies, the membranes were incubated with secondary antibodies that were conjugated with horseradish peroxidase. This incubation was carried out at room temperature for one hour. The horseradish peroxidase enzyme allows for the detection of the bound antibodies through a chemiluminescent reaction.

The protein bands on the membranes were then visualized using ECL detection reagents, a commercially available kit from Thermo Scientific, located in Waltham, Massachusetts. Finally, the intensity or density of the visualized bands was quantified using Image J software. To ensure the reliability and comparability of the results, the band densities of the target proteins were normalized to the levels of housekeeping proteins, such as glyceraldehyde-3-phosphate dehydrogenase or GAPDH, or Lamin B, which are proteins known to be expressed at relatively constant levels within the cells.

Nuclear fractionation

Cells were seeded in 10-cm dish and treated with PL at 0–40 μM for 24 h. Cells were lysed in hypotonic buffer solution (20 mM Tris (pH 7.4), 10 mM NaCl, 3 mM MgCl2) containing a protease inhibitor mixture. After addition of 10% Triton X-100, cell lysates were centrifuged at 650xg for 10 min at 4 °C. Pellets were resuspended in cell extraction buffer (100 mM Tris (pH 7.4), 100 mM NaCl, 1% Triton X-100, 10% glycerol, 0.1% SDS) containing a protease inhibitor mixture. The homogenates were centrifuged at 14,000xg for 30 min at 4 °C. The supernatants were collected as the nuclear fraction. Aliquots of nuclear were stored at −80 °C until use.

Statistical analysis

Data were expressed as mean ± standard error of the mean (SEM). Statistical significance was determined with SPSS-PASW statistics software version 18.0 for windows (SPSS, Chicago, IL) by one-way ANOVA and the groups were compared using Tukey test. A probability value p < 0.05 was considered statistically significant.

Results

Effect of PL on cancer cell proliferation

To determine if the substance PL has an effect on the growth and multiplication of A549 cells, a trypan blue dye exclusion test was conducted. In this assay, cells that had reached approximately 60% confluence in their culture vessels were treated with varying concentrations of PL, ranging from 0 to 40 micromolar, for a period of 24 hours. The results of this test indicated that PL significantly reduced the proliferation of these cells starting from a concentration of 5 micromolar. Furthermore, the extent of this reduction in cell proliferation was observed to be directly related to the concentration of PL applied, with higher concentrations leading to a greater inhibitory effect. Through analysis of the dose-response relationship, the half maximal inhibitory concentration, or IC50, of PL for A549 cell proliferation was calculated to be 27.3 micromolar. Based on the findings obtained from the trypan blue dye exclusion test, a range of PL concentrations spanning from 0 to 40 micromolar, which demonstrated an inhibitory effect on cell proliferation, were selected for use in the subsequent experimental investigations.

Effects of PL on cell cycle distribution

To examine the impact of the substance PL on the progression of cells through the cell cycle, the distribution of cells across the different phases of the cell cycle was analyzed using flow cytometry. Prior to this analysis, cells were treated with varying concentrations of PL, ranging from 0 to 40 micromolar, for a duration of 24 hours. The results obtained from this analysis revealed that the percentage of cells residing in the G1 phase of the cell cycle was significantly higher in cells treated with a 40 micromolar concentration of PL when compared to the untreated control group.

Specifically, the percentage of cells in the G1 phase in the control group was 65.82%, while in the group treated with 40 micromolar PL, it increased to 73.70%. Conversely, when cells were pretreated with NAC, the PL-induced increase in the proportion of cells in the G1 phase was diminished. Furthermore, the analysis showed that the percentage of cells in the S phase of the cell cycle was lower in cells treated with 40 micromolar PL compared to the control group. Taken together, these findings suggest that the observed reduction in cell proliferation caused by PL is likely mediated through the arrest of cells in the G1 phase of the cell cycle, preventing their progression into subsequent phases of cell division.

PL-induced ROS production in A549 cells

Reactive oxygen species are known to be significantly involved in the progression of the cell cycle. Notably, cancer cells typically exhibit elevated levels of reactive oxygen species, such as hydrogen peroxide, in comparison to their normal cellular counterparts. As a consequence of this inherent increase in basal ROS levels, cancer cells are often more vulnerable to further damage induced by additional reactive oxygen species. To investigate the generation of intracellular reactive oxygen species, specifically hydrogen peroxide, within the treated cells, a fluorogenic dye known as DCF-DA was employed. This dye becomes fluorescent upon oxidation in the presence of reactive oxygen species inside the cells, allowing for their detection and quantification. The intensity of the emitted fluorescent light serves as an indicator of the amount of reactive oxygen species produced within the cells.

In these experiments, cells were treated with varying concentrations of PL, ranging from 0 to 40 micromolar, for a duration of one hour. To validate the specificity of the observed effects, a control experiment was performed in which cells were pretreated with N-acetylcysteine, an antioxidant substance, at a concentration of 5 millimolar for one hour prior to the application of PL treatment at a concentration of 40 micromolar for one hour. The results obtained from these experiments demonstrated that PL significantly increased the production of reactive oxygen species within the cells at concentrations of 20 and 40 micromolar, when compared to the untreated control group. Furthermore, the pretreatment with NAC effectively inhibited the reactive oxygen species production induced by PL. These findings indicate that PL is capable of generating reactive oxygen species in cancer cells.

In addition to microscopic visualization, cells incubated with DCF-DA were also analyzed for reactive oxygen species production using a CytoFLEX flow cytometer. The results from this flow cytometric analysis corroborated the previous findings, showing a dose-dependent increase in reactive oxygen species production in cells with increasing concentrations of PL treatment. Moreover, the pretreatment of cells with NAC was shown to decrease the reactive oxygen species production induced by PL, further supporting the role of oxidative stress in the cellular response to PL.

PL-induced inhibition of cell proliferation and the role of oxidative stress

In order to investigate whether PL-induced oxidative stress is linked to cancer cell proliferation, trypan blue dye exclusion test was per- formed using an antioxidant, NAC. Cells with about 60% confluency were pretreated with NAC (5 mM, 1 h), followed by treatment of PL (40 μM, 24 h). PL significantly decreased cell proliferation up to 49%, compared to no treatment control. However, PL-induced inhibition of cell proliferation was significantly recovered by treatment of NAC. This data suggests that oxidative stress produced by PL plays a critical role in the proliferation of A549 cells.

PL-induced downregulation of cyclin D1, CDK4, CDK6 and Rb

In the cells of mammals, cyclins and cyclin-dependent kinases, or CDKs, are crucial regulators of various cellular processes, including the critical transition from the G1 phase to the S phase during the progression of the cell cycle. To gain a deeper understanding of the mechanisms underlying the PL-induced reduction in cell proliferation, the protein expression levels of key cell cycle-associated proteins, specifically cyclin D1, CDK4, CDK6, and retinoblastoma protein or Rb, were assessed using Western blot analysis.

In these experiments, cells were treated with varying concentrations of PL, ranging from 0 to 40 micromolar, for a period of 24 hours. The results of the Western blot analysis revealed that the protein expression levels of cyclin D1, CDK4, and CDK6 were significantly reduced in cells treated with PL at a concentration of 40 micromolar. Additionally, PL treatment at 40 micromolar also resulted in a significant decrease in the expression of phosphorylated retinoblastoma protein, denoted as p-Rb, when compared to the untreated control group. These findings collectively suggest that PL may induce an arrest in the cell cycle specifically at the transition point between the G1 and S phases in A549 cells. This arrest appears to be mediated by the reduction in the expression of essential cell cycle regulatory proteins, thereby hindering the cells’ ability to proceed from the growth phase (G1) to the DNA synthesis phase (S).

PL-induced intracellular signaling pathway in A549 cells

To pinpoint the intracellular signaling pathways that are involved in the PL-induced reduction of cell cycle-associated proteins, the phosphorylation status of Akt and mitogen-activated protein kinases, specifically p38 and extracellular signal-regulated kinases 1 and 2 (ERK1/2), was assessed in cells treated with PL. In these experiments, cells were exposed to PL at concentrations ranging from 0 to 40 micromolar for a duration of one hour. The results of these analyses indicated that PL treatment markedly decreased the phosphorylation of Akt starting at a concentration of 20 micromolar. Conversely, the phosphorylation of ERK1/2 was observed to increase in cells treated with PL, also starting at a concentration of 20 micromolar. At lower PL concentrations of 5 and 10 micromolar, only an insignificant decrease in Akt phosphorylation and a similarly insignificant increase in ERK1/2 phosphorylation were noted. The phosphorylation levels of p38, however, did not appear to be affected by PL treatment across the tested concentration range.

Furthermore, experiments involving pretreatment with the antioxidant N-acetylcysteine, at a concentration of 5 millimolar for one hour prior to PL treatment, revealed that NAC was able to reverse the PL-induced dephosphorylation of Akt. In contrast, NAC pretreatment led to a decrease in the PL-induced phosphorylation of ERK1/2. These findings collectively suggest that PL has the ability to modulate key intracellular signaling pathways, particularly Akt and ERK1/2. Moreover, the data indicate that the oxidative stress generated by PL plays a role in influencing the activity of these cell signaling pathways.

Modulation of NF-κB pathway by PL

Nuclear factor kappa B, commonly known as NF-κB, is a protein complex that plays a significant role in regulating the expression of various genes involved in cellular proliferation, including cyclin D1 and cyclin-dependent kinases. The activation of NF-κB can be assessed by observing the movement, or translocation, of its p65 subunit from the cytoplasm, the main fluid-filled space of a cell, into the nucleus, the cell’s control center containing genetic material. In the present investigation, cells were treated with PL for a duration of 24 hours to examine its potential influence on NF-κB activity. The results of these experiments showed that PL treatment led to a decrease in the translocation of the NF-κB p65 subunit into the nucleus. This finding suggests that while PL can induce oxidative stress and subsequently activate ERK1/2, this activation does not result in an increased activation of NF-κB. Instead, the observed dephosphorylation of Akt induced by PL appears to be the primary event leading to the deactivation of NF-κB.

To further confirm the critical role of Akt in the observed NF-κB activation state, cells were treated with LY294002, a specific pharmacological inhibitor that blocks the activity of Akt, at concentrations ranging from 15 to 30 micromolar. The data obtained from these experiments demonstrated that inhibiting Akt with LY294002 resulted in a reduction in the nuclear translocation of the NF-κB p65 subunit, mimicking the effect of PL treatment. However, when cells were treated with PD98059, a specific inhibitor of ERK1/2, there was no significant influence observed on the activation of NF-κB. These results collectively confirm that the inactivation of NF-κB induced by PL is primarily mediated through the dephosphorylation of Akt, rather than through the activation of ERK1/2 signaling.

Role of Akt and ERK1/2 in PL-induced downregulation of cell cycle- associated proteins

Given the observation that PL can alter the activity of both Akt and ERK1/2 signaling pathways, as well as the expression levels of key cell cycle-associated proteins such as cyclin D1, CDK4, CDK6, and phosphorylated retinoblastoma protein, further investigation was conducted to elucidate the specific roles of these signaling molecules in A549 cells. To this end, cells were treated with specific pharmacological inhibitors targeting Akt (LY294002) and ERK1/2 (PD98059) for a duration of 24 hours, and the expression of these proteins was monitored. Additionally, a trypan blue dye exclusion test was performed to assess cell viability in cells treated with each of these pharmacological inhibitors.

The treatment of cells with the Akt inhibitor, LY294002, resulted in a significant decrease in cell proliferation in a manner that was dependent on the concentration of the inhibitor used. Furthermore, the protein expression levels of the aforementioned cell cycle-associated proteins, namely cyclin D1, CDK4, CDK6, and phosphorylated retinoblastoma protein, were also reduced in cells treated with LY294002 in a dose-dependent manner. These findings strongly corroborate the notion that the deactivation of Akt is a critical event in the process by which PL inhibits the proliferation of cancer cells.

In contrast, the treatment of cells with the ERK1/2 inhibitor, PD98059, did not have a significant effect on the proliferation of these cells. Moreover, the protein expression levels of the cell cycle-associated proteins that were previously shown to be decreased by PL treatment were not further influenced by the application of the ERK1/2 inhibitor. These results collectively indicate that the Akt signaling pathway, and not the ERK1/2 pathway, plays a primary role in mediating the PL-induced inhibition of proliferation in A549 cells.

Discussion

Piperlongumine, often abbreviated as PL, is an alkaloid compound that is naturally derived from the long pepper plant, scientifically known as Piper longum L. This compound has been identified as possessing a wide range of biological activities, notably including anticancer effects. A significant aspect of PL’s anticancer properties is its ability to selectively target and eliminate various types of cancer cells while exhibiting minimal toxicity towards normal, healthy cells. Apoptosis, or programmed cell death, has been established as a key mechanism through which PL mediates the death of cancer cells. However, other potential mechanisms by which PL might inhibit cancer development and progression, particularly in the context of lung cancer cells such as the A549 cell line, have not been fully elucidated. Therefore, this study aimed to investigate whether PL could inhibit the proliferation of A549 lung cancer cells by altering the expression of proteins that are associated with the regulation of the cell cycle.

In this study, the effects of PL on the proliferation of A549 cells were specifically examined. To achieve this, trypan blue dye exclusion assays were performed. In these experiments, cells that had reached a confluence of approximately 50 to 60% were intentionally treated with PL. This specific cell density was chosen because the cells at this stage exhibit active cell division and are expected to reach 100% confluence within a 24-hour period under normal growth conditions. Consequently, during this 24-hour treatment window, the rate of cell proliferation would be highly sensitive to the presence or absence of PL in the culture medium.

The results of these assays demonstrated that PL reduced cell proliferation starting at a concentration of 5 micromolar, and this inhibitory effect was observed to increase in a dose-dependent manner, meaning that higher concentrations of PL led to a greater reduction in cell proliferation. These findings are consistent with previously reported results in A549 cells, where a 10 micromolar concentration of PL resulted in approximately 60% cell confluence after 24 hours, in contrast to the control group that reached full confluence.

Furthermore, flow cytometry analysis conducted in this study revealed an increased number of cells in the G1 phase of the cell cycle when the cells were treated with PL. This observation suggests that the reduction in cell proliferation induced by PL is at least partially attributable to an arrest of the cell cycle in the G1 phase, preventing the cells from progressing to the DNA synthesis (S) phase and subsequent cell division. Interestingly, in this flow cytometry analysis, a significant population of cells in the sub-G1 phase, which is typically indicative of apoptotic cells with fragmented DNA, was not detected.

This absence is likely due to the removal of most apoptotic cells during the washing steps with phosphate-buffered saline that were performed when collecting the cells using trypsinization. However, previous research has reported a marked increase in apoptotic cells in A549 cells treated with PL. Although apoptotic cell death was not directly measured in the current study, these prior findings suggest that apoptosis also contributes to the decreased cell number observed in our trypan blue dye exclusion tests following PL treatments.

The progression of the cell cycle and the overall rate of cell proliferation are governed by a complex network of cellular molecules. A common characteristic observed in the process of carcinogenesis is the upregulation of cellular proliferation and the constitutive, or persistent, activation of the transcription factor NF-κB. This study specifically investigated the mechanisms underlying cell proliferation, with a particular focus on the modulation of cell cycle-associated proteins by PL. Previous research has established that cell proliferation is regulated by various cellular signaling pathways and molecules, including the mitogen-activated protein kinase (MAPK) pathway, the Akt signaling pathway, and cyclins.

The cell cycle is a carefully orchestrated series of events, and the transition from the G1 phase to the S phase is a critical step in this process, determining whether a cell will proceed to duplicate its DNA and eventually divide. Cyclin D1, in conjunction with its catalytic partners cyclin-dependent kinase 4 (CDK4) and cyclin-dependent kinase 6 (CDK6), plays a key regulatory role in the G1 phase of the cell cycle. To examine the effect of PL on the cell cycle in A549 cells, the expression levels of these cell cycle-associated proteins were investigated using Western blot analysis.

The protein expression levels of cyclin D1, CDK4, and CDK6 have been previously and successfully used as markers to assess cell cycle progression in cancer cells. The results of our Western blot analysis showed that PL significantly decreased the expression of cyclin D1, CDK4, and CDK6 in A549 cells. Our data indicate that these reductions in protein levels are associated with the inhibition of cell proliferation observed in the trypan blue exclusion experiments. Similar findings have been reported in other types of cancer cells, where PL inhibited cancer cell proliferation by arresting cells in the G1 phase in human breast cancer cells (MDA-MB231) and hepatocellular carcinoma cells (HCC).

The phosphorylation of the retinoblastoma protein, known as Rb, is a critical regulatory step within the cell cycle. Rb functions as a key component of the G1 checkpoint, effectively acting as a molecular gatekeeper that prevents cells from prematurely entering the S phase, the phase of DNA synthesis. Furthermore, Rb plays a role in inhibiting cell proliferation by inducing cell cycle arrest specifically at the G1 phase. It has been established that the cyclin D1 protein, in complex with its kinase partners CDK4 and CDK6, phosphorylates Rb during the transition from the G1 phase to the S phase of the cell cycle.

Our experimental data revealed a decrease in the phosphorylation of Rb in cells treated with a 40 micromolar concentration of PL. This reduction in Rb phosphorylation is likely a direct consequence of the observed decrease in the expression levels of cyclin D1, CDK4, and CDK6 in these treated cells. Consequently, our findings strongly suggest that the PL-induced inhibition of cell proliferation is intricately linked to the altered cellular levels of these crucial cell cycle regulatory proteins.

Reactive oxygen species, or ROS, play a significant role as signaling molecules that modulate the activity of various proteins controlling fundamental cellular processes such as cell proliferation, migration, and apoptosis, or programmed cell death. However, excessive accumulation of ROS can lead to oxidative damage within cells and induce cell cycle arrest, particularly in cancer cells. Consistent with this, previous research has shown that andrographolide, a compound derived from traditional herbal medicine, arrested the cell cycle in HepG2 liver cancer cells by causing an accumulation of excessive ROS, primarily hydrogen peroxide.

In agreement with these prior findings, our data demonstrated that PL treatment markedly increased the production of ROS in A549 cells at concentrations ranging from 20 to 40 micromolar. The importance of ROS production in the observed inhibition of cell proliferation was further confirmed in subsequent experiments where pretreatment of cells with N-acetylcysteine, an antioxidant compound, effectively negated both the PL-induced ROS production and the inhibition of cell proliferation. Our data thus support previous findings indicating that PL can target intracellular stress response pathways, leading to ROS accumulation as a mechanism to selectively kill cancer cells.

The cell cycle arrest induced by ROS is known to be mediated by the modulation of intracellular signaling molecules, including ERK1/2, Akt, and NF-κB. In the current study, we observed that Akt was significantly dephosphorylated in A549 cells treated with 20 to 40 micromolar PL, whereas ERK1/2 exhibited increased phosphorylation in a dose-dependent manner in response to PL treatment. Notably, pretreatment of cells with NAC reversed both the PL-induced dephosphorylation of Akt and the phosphorylation of ERK1/2.

These findings indicate that PL can selectively modulate key intracellular signaling molecules, specifically Akt and ERK1/2, which in turn may influence the levels of cell cycle regulatory proteins and ultimately affect cell proliferation. Similar to PL, sorafenib, a drug used in the treatment of renal cell carcinoma, has been shown to decrease the phosphorylation of Akt, and NAC was able to rescue cells from this sorafenib-induced Akt dephosphorylation.

In fact, it has been reported that phosphorylated Akt promotes cell survival pathways and is also associated with cancer cell progression in various human carcinomas, including lung cancer. Our data demonstrated that Akt, but not ERK1/2, is primarily associated with the expression of cell cycle-associated proteins in A549 cells. Furthermore, our findings suggest that the ROS produced in response to PL treatment acts as a modulator for the Akt signaling pathway.

The Akt signaling pathway is known to be involved in cell survival and proliferation across a wide range of cancer cell types. For instance, PL has been shown to suppress the phosphorylation of Akt, which subsequently led to the inhibition of cyclin D1 expression in MDA-MB-231 breast cancer cells. Akt is also known to be associated with the activation of NF-κB. Tumor samples obtained from human cancer patients have shown high levels of NF-κB activation. Consequently, the activation of NF-κB plays a critical role in protecting cells by promoting cell proliferation and suppressing cell apoptosis.

Consistent with this, previous research has shown that ganoderma lucidum, an Asian mushroom, suppressed the phosphorylation of Akt, resulting in the attenuation of NF-κB activation in MDA-MB-231 cells. Moreover, the downregulation of NF-κB has been shown to decrease the expression of cyclin D1 and CDK4. Similar to these prior reports, our data demonstrated that PL significantly decreased the activation of NF-κB in A549 cells. The involvement of Akt in this process was confirmed in experiments where cells treated with LY294002, a specific inhibitor of Akt, exhibited decreased nuclear translocation of NF-κB.

These data indicate that the PL-induced dephosphorylation of Akt can lead to the downregulation of NF-κB activation in A549 cells. Furthermore, we determined a correlation between Akt phosphorylation levels and the expression of cell cycle-associated proteins in A549 cells. Treatment of cells with the Akt inhibitor LY294002 significantly decreased cancer cell proliferation and the expression of key cell cycle-associated proteins, including cyclin D1, CDK4, CDK6, and phosphorylated Rb, in a dose-dependent manner.

We also investigated the potential involvement of ERK1/2 signaling in the PL-induced decrease of cell proliferation and cell cycle protein expression, given that PL treatment increased ERK1/2 phosphorylation. However, in experiments using PD98059, an inhibitor specific for ERK1/2, the PL-induced decrease in cell proliferation and cell cycle protein expression was not affected by the inhibition of ERK1/2. These findings suggest that the observed cellular responses to PL in A549 cells are primarily mediated through the Akt-NF-κB signaling pathway, rather than the ERK1/2 pathway. Previous research has demonstrated that the inhibition of Akt can decrease the expression of cell cycle-associated proteins, such as cyclin D1, CDK4, and CDK6, in human ovarian cancer cells.

Additionally, similar to the effects of PL, IQDMA, a synthesized quinolone derivative, has been shown to phosphorylate ERK1/2 and induce cell cycle arrest in A549 cells. Importantly, the IQDMA-induced cell cycle arrest was not affected by a pharmacological inhibitor of ERK1/2, consistent with our findings regarding PL. These results suggest that while ERK1/2 may be involved in other cellular processes, such as the modulation of pro-apoptotic genes, it does not appear to play a primary role in the regulation of cell cycle protein expression in response to PL in A549 cells.

Our results contribute to the growing body of evidence linking plant-derived compounds with anti-carcinogenic properties. Other known bioactive compounds found in medicinal herbs and plants, such as ursolic acid and epigallocatechin gallate (EGCG), have demonstrated antiproliferative effects through mechanisms similar to those observed with PL. Ursolic acid has been shown to inhibit cell proliferation by blocking cell cycle progression through G1 phase arrest, which is achieved by decreasing the levels of cell cycle-associated proteins like cyclin D1, cyclin D2, CDK4, and CDK6.

Additionally, ursolic acid upregulated proapoptotic molecules and downregulated antiapoptotic molecules in A549 cells. Similarly, Kotomolide A, a plant-derived substance, arrested cell cycle progression in the G2/M phase by activating p53 in A549 cells. In other studies, epigallocatechin gallate, a component of green tea, inhibited cell proliferation and Akt phosphorylation in A549 cells. Furthermore, flavokavain C, isolated from Kava root, arrested the cell cycle by suppressing Akt phosphorylation and inducing ERK1/2 phosphorylation in the HCT116 colon carcinoma cell line. Therefore, natural products derived from herbal or dietary plants represent important sources of potential therapeutic substances for cancer treatment.

In conclusion, our data suggest that PL can inhibit the proliferation of A549 lung cancer cells by suppressing the expression of key cell cycle proteins, including cyclin D1, CDK4, CDK6, and phosphorylated Rb. During this antiproliferative process initiated by PL treatment, we identified several important cellular events, such as the production of reactive oxygen species, decreased Akt phosphorylation, and the deactivation of NF-κB. Our findings provide in vitro evidence that PL possesses significant antiproliferative properties in human lung carcinoma cells and could potentially serve as a therapeutic agent in the future.