Pifithrin-μ is efficacious against non-small cell lung cancer via inhibition of heat shock protein 70
Abstract
Heat-shock protein 70, frequently abbreviated as Hsp70, is a widely recognized and crucial molecular chaperone within cellular biology, predominantly known for its potent pro-survival capabilities. This essential protein plays a fundamental role in maintaining cellular homeostasis by facilitating the proper folding and assembly of newly synthesized polypeptides, assisting in the refolding of misfolded proteins, and targeting damaged proteins for degradation. In the context of pathology, Hsp70 is regrettably observed to be aberrantly or overexpressed in a diverse array of human malignancies, where its heightened presence often contributes to the survival and proliferation of cancerous cells, rendering them more resistant to conventional therapeutic interventions.
The small molecule identified as 2-phenylethyenesulfonamide, also widely recognized by its alternative designation pifithrin-μ (PES), has garnered significant attention within the oncology field as a specific inhibitor of Hsp70. This compound has demonstrated considerable promise by exhibiting notable antitumor activities across a broad spectrum of various cancer cell lines. However, despite these encouraging findings in other cancer types, there has been a significant dearth of knowledge regarding its precise effects on non-small cell lung cancer (NSCLC) cell lines, a particularly aggressive and prevalent form of malignancy.
Consequently, the primary objective of this comprehensive study was to systematically investigate the therapeutic potential of PES against human NSCLC, specifically utilizing two representative cell lines, A549 and H460, which are commonly employed as *in vitro* models for this disease. Beyond merely observing its effects, a key aim was to meticulously explore the potential underlying molecular mechanisms through which PES exerts its anticancer actions. To quantitatively assess cellular proliferation, a cell viability assay employing the Cell Counting Kit-8 (CCK-8) method was performed. The results from this assay unequivocally demonstrated that PES effectively inhibited the proliferation of both A549 and H460 cells in a manner that was both dependent on the administered dose and the duration of treatment, with higher concentrations and longer exposure times leading to greater growth inhibition.
Beyond proliferation, the metastatic potential of cancer cells is a critical determinant of patient prognosis. To evaluate the impact of PES on cellular motility, a wound healing assay and a Transwell migration assay were meticulously conducted. The outcomes from both these complementary techniques consistently indicated that PES significantly suppressed the migratory capabilities of both A549 and H460 cells, suggesting a potential role in inhibiting metastasis.
To further elucidate the cellular processes affected by PES, flow cytometry analysis was performed. These detailed investigations revealed that PES treatment led to a distinct arrest of cancer cells in the G0/G1 phase of the cell cycle. This G0/G1 phase arrest effectively halts cell division, preventing the cells from progressing towards replication, thereby contributing to the observed antiproliferative effect. Furthermore, the flow cytometry results, in conjunction with caspase activity assays, demonstrated that PES effectively induced apoptosis, a form of programmed cell death, in both A549 and H460 cells. This apoptotic induction was confirmed to proceed via a caspase-dependent pathway, indicating the activation of key enzymatic cascades responsible for dismantling cellular components.
To unravel the molecular underpinnings of these observed effects, Western blotting analyses were performed. These analyses provided crucial insights, indicating that PES treatment effectively inhibited the phosphorylation of both AKT and ERK. Both AKT and ERK are pivotal signaling proteins involved in crucial cellular processes such as cell survival, proliferation, and differentiation, and their reduced phosphorylation typically signifies the dampening of pro-survival pathways. In addition to these findings, Western blotting also revealed that treatment with PES led to an increased expression of death receptor 4 (DR4) and death receptor 5 (DR5). These death receptors are integral components of the extrinsic apoptotic pathway, serving as cell surface conduits for apoptotic signals, and their upregulation suggests a sensitization of cancer cells to pro-apoptotic stimuli.
To directly confirm that the observed antitumor effects of PES were indeed mediated specifically through its interaction with Hsp70, further experiments were conducted involving genetic manipulation of Hsp70 levels. When Hsp70 was deliberately overexpressed in A549 cells, this overexpression significantly attenuated the growth inhibitory efficiency of PES, suggesting that higher levels of Hsp70 could counteract PES’s effects. Conversely, when Hsp70 expression was intentionally reduced or knocked down in A549 cells using specific small interfering RNA (siRNA), the sensitivity of these cells to PES-induced cell growth inhibition was markedly enhanced. These compelling reciprocal findings provide strong evidence that the inhibitory effect of PES on cancer cell proliferation is specifically mediated through an Hsp70-dependent mechanism, solidifying its role as a targeted Hsp70 inhibitor.
Beyond monotherapy, the potential for combination therapy was also explored. The study revealed that when PES was combined with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a potent synergistic effect was observed. This combination therapy significantly enhanced both the inhibition of cell proliferation and the induction of apoptosis in both A549 and H460 cells, highlighting a promising therapeutic strategy for overcoming resistance.
Finally, to translate these promising *in vitro* findings into a more clinically relevant context, the antitumor activity of PES was evaluated in an *in vivo* mouse xenograft model of lung cancer, established by subcutaneously implanting A549 cells into immunodeficient mice. In this preclinical model, PES treatment robustly displayed significant inhibitory effects on the growth of the established tumors, demonstrating its efficacy in a living organism. Collectively, all these comprehensive findings from both *in vitro* and *in vivo* experiments strongly suggest that PES possesses substantial antitumor activity against human non-small cell lung cancer. Consequently, based on its multifaceted mechanisms of action and demonstrated efficacy, PES emerges as a highly promising therapeutic agent for future consideration in the treatment of NSCLC.
Introduction
Lung cancer represents one of the most pervasive and unfortunately, one of the most fatal types of cancers globally, imposing an immense burden on public health systems worldwide. This devastating malignancy is responsible for approximately 20% of all cancer-related deaths, underscoring its significant contribution to global mortality statistics. Broadly, lung cancer is categorized into two principal types: small cell lung cancer and non-small cell lung cancer (NSCLC). Among these, NSCLC constitutes the vast majority of cases, accounting for approximately 80% of all diagnosed lung cancer instances. Despite advancements in medical science, NSCLC tragically leads to the highest mortality rates among lung cancers, characterized by a relatively poor 5-year survival rate, a stark indicator of the challenges in its effective management.
While significant strides have been made in the development of surgical techniques and various chemotherapeutic regimens for the treatment of lung cancer, the overall prognosis for patients afflicted with NSCLC remains considerably bleak. This poor prognosis is largely attributable to several formidable challenges, including the inherent toxicity often associated with conventional chemotherapy drugs, leading to severe side effects that compromise patient quality of life. Furthermore, a persistently high incidence of disease recurrence following initial treatment contributes significantly to the low long-term survival rates. Indeed, despite therapeutic progress, the 5-year survival rate for NSCLC patients remains distressingly low, hovering around 15%. This grim reality underscores the urgent and pressing need for the exploration and development of novel therapeutic strategies and pharmacological agents. The ideal next-generation drugs would not only demonstrate superior efficacy in combating NSCLC but would also be characterized by fewer debilitating side effects, thereby improving both patient outcomes and their overall quality of life during treatment.
The heat shock protein (Hsp) family constitutes a highly conserved and universally present group of molecular chaperones, proteins indispensable for maintaining cellular integrity and function. These remarkable proteins perform vital roles in the cell, primarily by facilitating the proper folding of newly synthesized proteins, assisting in the refolding of proteins that have become denatured or misfolded due to stress, and orchestrating the efficient transportation of proteins to their correct subcellular destinations. Beyond their fundamental roles in protein quality control, Hsps are also critically recognized as potent inhibitors of apoptosis, or programmed cell death, thereby promoting cell survival. The expression levels of Hsps typically undergo a dramatic increase within cells when they are subjected to various forms of cellular stress, including but not limited to elevated temperatures (heat shock), conditions of oxygen deprivation (hypoxia), and exposure to a wide array of cytotoxic agents, such as those used in chemotherapy. This inducible upregulation of Hsps serves as a crucial protective mechanism, safeguarding cells from injury and promoting their resilience in adverse environments.
Among the various members of the Hsp family, Hsp70 has attracted particular scrutiny in the context of cancer. Compelling evidence from numerous studies has consistently reported a direct association between Hsp70 overexpression and a wide spectrum of human malignancies. This heightened expression of Hsp70 in cancer cells is not merely an incidental finding; rather, accumulating results have specifically highlighted that, when compared to healthy individuals, Hsp70 is significantly overexpressed in both the serum and tissue samples obtained from patients diagnosed with NSCLC. This aberrant overexpression in NSCLC is particularly concerning because it has been further linked to adverse patient prognosis and, critically, to the development of resistance to conventional chemotherapy regimens. This resistance mechanism is believed to stem from Hsp70′s anti-apoptotic properties, which allow cancer cells to evade death signals induced by therapeutic agents. These observations have profoundly influenced the direction of cancer research, leading to the compelling hypothesis that selectively targeting or depleting Hsp70 in lung cancer cells could represent a highly promising and innovative therapeutic approach for cancer treatment. Notably, studies have shown that selective depletion of Hsp70 in lung cancer cells leads to apoptotic cell death, while exhibiting a favorable safety profile by not inducing similar detrimental effects in normal lung cells, thus highlighting its potential as a selective anticancer target.
In recent years, the small molecule designated as 2-phenylethynesulfonamide, widely recognized by its alternative designation pifithrin-μ (PES), has been identified as a specific and potent inhibitor of stress-inducible Hsp70. This compound has demonstrated a remarkable ability to selectively induce tumor cell death while concurrently exhibiting markedly reduced toxicity towards non-transformed, healthy cells, suggesting a favorable therapeutic window. Its mechanism of action is believed to involve the impairment of several pivotal survival pathways critical for cancer cell viability, primarily achieved through the disruption of the crucial HSP70/HSP90 chaperone system, which is frequently exploited by cancer cells for their aberrant growth. Further studies have illuminated additional mechanisms, with data indicating that PES can induce immunogenic cell death, a type of cell death that elicits an immune response against the tumor, specifically via the release of lysosomal cathepsin D in primary effusion lymphoma. Moreover, PES has demonstrated a valuable synergistic effect when combined with other anticancer agents. For instance, it has been shown to enhance the antitumor activities of 17-AAG, an Hsp90 inhibitor, against acute leukemia and bladder cancer cells. Beyond chemical synergy, PES has also synergistically enhanced the antitumor activity of hyperthermia, a physical therapeutic modality, against prostate cancer cells. However, despite these diverse and encouraging findings across various cancer types, there remains a notable gap in the scientific literature regarding the precise effect of PES on human lung cancer cells. Specifically, no comprehensive study had been conducted to evaluate whether PES exhibits a chemosensitizing effect on human lung cancer cells, which is its ability to make cancer cells more susceptible to other forms of therapy.
In light of these critical knowledge gaps, the present investigation was specifically undertaken to thoroughly explore the capacity of PES to inhibit the proliferation of non-small cell lung cancer cell lines, both in controlled *in vitro* laboratory settings and within a complex *in vivo* animal model. A further, equally important objective was to meticulously unravel the underlying molecular mechanisms responsible for its observed anticancer effects. Additionally, we aimed to systematically evaluate the synergistic antitumor effect of PES when administered in combination with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a promising pro-apoptotic agent. Our comprehensive results provide compelling evidence that PES can effectively inhibit both cell proliferation and cell migration in human NSCLC cells, demonstrating its multifaceted anticancer properties. Furthermore, our findings reveal that PES is capable of inducing a specific G0/G1 phase cell cycle arrest, thereby halting cell division, and promoting cell apoptosis through a caspase-dependent pathway, signifying its ability to trigger programmed cell death. Crucially, the specificity of PES’s action via Hsp70 was strongly supported by our genetic manipulation experiments: overexpression of Hsp70 in A549 cells notably attenuated the inhibitory effect of PES on cell proliferation, while conversely, the targeted knockdown of Hsp70 using small interfering RNA in A549 cells demonstrated a potent synergistic effect on cell proliferation inhibition. Our molecular analyses further indicated that beyond its direct Hsp70 inhibition, PES contributes to its antitumor effects through the reduction of phosphorylated AKT (p-AKT) and phosphorylated ERK (p-ERK), key signaling molecules involved in cell survival and growth. Moreover, PES was found to sensitize NSCLC cell lines to TRAIL-induced cell proliferation inhibition and apoptosis, a synergistic effect achieved, at least in part, via the upregulation of death receptor 4 (DR4) and death receptor 5 (DR5), rendering the cells more susceptible to apoptotic signals. Finally, the translational relevance of our findings was substantiated by *in vivo* experiments, where lung cancer xenografts in nude mice were efficiently and significantly inhibited by PES treatment, confirming its therapeutic efficacy in a living organism.
Materials and Methods
Cell Lines and Reagents
For the rigorous execution of the experimental studies, the human non-small cell lung cancer (NSCLC) cell lines, A549 and H460, which are well-established and widely utilized models in lung cancer research, were specifically purchased from the American Type Culture Collection (Manassas, Virginia, USA), ensuring their authenticity and genetic integrity. These cell lines were meticulously maintained under optimal growth conditions to ensure consistency and viability throughout the experimental period. Their culture involved the use of Dulbecco’s Modified Eagle Medium (DMEM), a foundational nutrient-rich basal medium, which was comprehensively supplemented with 10% fetal bovine serum (FBS), a vital source of growth factors and nutrients obtained from Gibco, Gaithersburg, Maryland, USA, essential for robust cellular proliferation. The cells were incubated at a precisely controlled temperature of 37 degrees Celsius within a humidified atmosphere containing 5% carbon dioxide, conditions designed to mimic the physiological environment conducive for mammalian cell growth. The key compound under investigation, the heat-shock protein 70 (Hsp70) inhibitor known as PES (2-phenylethyenesulfonamide), also referred to as pifithrin-μ, was procured from Calbiochem (San Diego, California, USA), ensuring its specific inhibitory activity. Recombinant tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a crucial protein involved in inducing apoptosis, was obtained from Invitrogen (Carlsbad, California, USA). To prepare working solutions for experimental use, PES and recombinant TRAIL were dissolved in specific carriers; PES was solubilized in dimethyl sulfoxide (DMSO), acquired from Sigma, St. Louis, Missouri, USA, while recombinant TRAIL was dissolved in phosphate-buffered saline (PBS) containing 0.1% (w/v) bovine serum albumin (BSA). The use of these solvents and stabilizers ensured the proper delivery and activity of the compounds in cellular assays.
Plasmids and Cell Transfection
To investigate the specific role of Hsp70 in mediating the effects of PES, genetic manipulation techniques involving the overexpression or knockdown of Hsp70 were employed. The plasmid designated pSG5-Hsp70, which carries the genetic code for the overexpression of Hsp70, was generously provided by Professor X. Sun from the School of Basic Medical Sciences, Wuhan University, Wuhan, China. This plasmid allowed for the controlled increase of Hsp70 protein levels within the cells. Conversely, to achieve the targeted reduction or knockdown of Hsp70 expression, specific Hsp70 small interfering RNA (siRNA) and a non-targeting control siRNA were obtained from GenePharma (GenePharma, Shanghai, China). These siRNAs are designed to specifically bind to and degrade Hsp70 mRNA, thereby preventing its translation into protein. For transient overexpression experiments, A549 cells were meticulously transfected with the pSG5-Hsp70 plasmid using the X-tremeGENE HP DNA Transfection Reagent, a highly efficient reagent from Roche (Basel, Switzerland), following the detailed instructions provided by the manufacturer. This transient transfection method ensures that the cells temporarily express elevated levels of Hsp70. Similarly, for gene knockdown experiments, A549 cells were transiently transfected with either the Hsp70 siRNA or the control siRNA utilizing Oligofectamine, a widely used transfection reagent from Invitrogen, according to the manufacturer’s precise guidelines. This approach allowed for the temporary reduction of endogenous Hsp70 protein levels to assess its impact on PES sensitivity.
Cell Viability Analysis
The quantitative assessment of cell viability, a fundamental measure of the impact of test compounds on cellular health and proliferation, was meticulously determined using the Cell Counting Kit-8 (CCK-8) assay, a robust and reliable colorimetric method supplied by Dojindo Laboratories, Kumamoto, Japan. The procedure commenced with the precise seeding of A549 and H460 cells into 96-well plates. Cells were plated at a standardized density of 5 x 10^3 cells per well, contained within 100 microliters of culture medium, and allowed to adhere overnight to ensure stable cell attachment. Following this initial plating, the cells were subjected to treatment with various indicated concentrations of PES, ranging from 2.5 to 40 micromolar, for two distinct durations: 24 hours and 48 hours. This dose- and time-dependent treatment regimen allowed for a comprehensive evaluation of PES’s antiproliferative effects. After the specified treatment period, 10 microliters of the CCK-8 tetrazolium substrate solution were carefully added to each well of the plate. This substrate is bioreduced by metabolically active cells into a yellow formazan dye, which is soluble in the cell culture medium. The plates were then incubated at 37 degrees Celsius for 1 hour, allowing sufficient time for the enzymatic conversion of the substrate. Subsequently, the optical density (OD) of each well was precisely measured at a wavelength of 450 nm using a microplate reader, specifically the EXL800 model from BioTek, Winooski, Vermont, USA. The absorbance value obtained is directly proportional to the number of viable cells in each well. To ensure the statistical reliability and reproducibility of the results, each experimental condition was performed in triplicate, meaning three independent wells were assessed, and the entire experiment was repeated independently at least three times, thereby providing robust data for analysis.
Wound Healing Assay
To evaluate the inhibitory effect of PES on the migratory capabilities of human NSCLC cells, a well-established *in vitro* assay known as the wound healing assay was performed. This assay directly assesses the collective movement of cells into a cell-free area, mimicking cell migration during wound repair or, in the context of cancer, metastatic dissemination. The procedure began by seeding A549 and H460 cells at a density of 5 x 10^5 cells into 6-well plates and allowing them to grow to confluence overnight, forming a uniform monolayer. Once confluent, a sterile 10-microliter pipette tip was carefully used to create a linear “wound” or scratch across the cell monolayer in each well, creating a cell-free gap. After creating the wounds, the cells were gently washed with phosphate-buffered saline (PBS) to remove any detached cells or debris from the scratch area. Subsequently, the culture medium was replaced with fresh medium, either containing or lacking PES, depending on the experimental group. The cells were then incubated for an additional 48 hours, during which time untreated cells would typically migrate to close the wound. Throughout this incubation period, the progress of wound closure was visually monitored and documented using an inverted microscope. By comparing the extent of wound closure in PES-treated cells versus control cells, the inhibitory effect of the compound on cellular migration could be qualitatively and semi-quantitatively assessed.
Transwell Migration Assay
To further and more quantitatively confirm the suppressive effect of PES on cell migration, independent of potential proliferation effects, a Transwell migration assay was rigorously conducted. This assay utilizes specialized Transwell chambers, procured from Corning, New York, NY, USA, which consist of an upper chamber separated from a lower chamber by a polycarbonate membrane. This membrane is precisely engineered with 8-micrometer polyester membrane filter pores, allowing for the passage of migrating cells but preventing passive diffusion of non-motile cells. Prior to the experiment, cells were starved for 24 hours to minimize proliferation and maximize their migratory responsiveness to a chemoattractant. Following starvation, 1 x 10^5 cells were carefully seeded into the upper chamber of the Transwell apparatus, suspended in 100 microliters of serum-free medium, which lacks growth factors that could promote proliferation or random movement. Conversely, the bottom chamber contained 500 microliters of medium supplemented with 10% fetal bovine serum (FBS), which served as a potent chemoattractant, creating a chemotactic gradient to stimulate cell migration through the pores. After an incubation period of 48 hours at 37 degrees Celsius, cells that had successfully migrated through the membrane pores and adhered to the lower surface of the membrane were meticulously fixed and stained. The stained cells were then captured using microscopy, and their numbers were quantified, providing a direct and quantitative measure of cell migratory capability. By comparing the number of migrated cells in PES-treated groups to control groups, the inhibitory effect of PES on cell migration was confirmed, providing strong evidence for its anti-metastatic potential.
Flow Cytometry Analysis
Flow cytometry, a powerful cell analysis technique, was employed to comprehensively assess two critical cellular processes modulated by PES: cell cycle arrest and the induction of apoptosis. For these analyses, A549 and H460 cells were seeded at a density of 2 x 10^5 cells per well in 6-well plates and subsequently treated with either a vehicle control solution or 20 micromolar of PES for a duration of 24 hours.
For the analysis of cell cycle distribution, the treated cells were meticulously collected and fixed overnight at -20 degrees Celsius using 500 microliters of 70% ethanol. This fixation step permeabilizes the cell membranes and preserves cellular DNA content, crucial for cell cycle assessment. Following fixation, the cells were washed three times with cold phosphate-buffered saline (PBS) to remove residual ethanol and cellular debris. The cell cycle distribution was then precisely determined using a Cell Cycle Detection kit, obtained from Multisciences, Hangzhou, China, strictly adhering to the manufacturer’s detailed instructions. This kit typically involves staining DNA with a fluorescent dye, allowing flow cytometry to quantify the DNA content of individual cells and thereby determine the proportion of cells in each phase of the cell cycle (G0/G1, S, G2/M). The analysis focused on identifying any accumulation of cells in specific cell cycle phases, such as G0/G1 phase cell cycle arrest, which indicates a block in cell proliferation.
For the analysis of apoptosis induction, A549 and H460 cells subjected to PES or vehicle control treatment were similarly collected. Cell apoptosis was then detected using the Annexin V-FITC/propidium iodide (PI) Apoptosis Detection kit, also from Multisciences, following the manufacturer’s instructions. This dual-staining method is widely used to distinguish between viable, early apoptotic, late apoptotic, and necrotic cells. Annexin V-FITC specifically binds to phosphatidylserine, a lipid that translocates to the outer leaflet of the cell membrane during early apoptosis. Propidium iodide (PI), a DNA-intercalating dye, is generally excluded from live cells but can enter cells with compromised membrane integrity, characteristic of late apoptosis or necrosis. By analyzing the differential uptake of these two fluorescent dyes via flow cytometry, the percentage of cells undergoing apoptosis in response to PES treatment could be accurately quantified, providing direct evidence for its pro-apoptotic effects.
Caspase-3 Activity Assay
To precisely determine whether the observed induction of apoptosis by PES occurred through a caspase-dependent mechanism, the activity of caspase-3, a pivotal executioner caspase, was quantified using specific assay kits obtained from Beyotime, Shanghai, China. The experimental procedure involved treating human NSCLC cells (A549 and H460) with either PES or TRAIL, or a combination of both, for a 24-hour period. Following treatment, the cells were meticulously washed three times with cold phosphate-buffered saline (PBS) to remove any residual compounds and media components. The washed cells were then subjected to lysis buffer treatment on ice for 30 minutes, a step designed to effectively break open the cells and release their intracellular contents, including active caspases. After lysis, the cell lysates were centrifuged at 12,000 g for 10 minutes at 4 degrees Celsius to pellet cellular debris, yielding a clear supernatant containing the cellular proteins. A precise aliquot of 10 microliters of this cell lysate supernatant was then combined with 80 microliters of assay buffer and 10 microliters of the specific caspase-3 substrate, Ac-DEVD-pNA, in each well of a 96-well plate. Ac-DEVD-pNA is a chromogenic substrate that is specifically cleaved by active caspase-3, releasing para-nitroaniline (pNA), a yellow compound. The samples were then further incubated at 37 degrees Celsius for 12 hours, allowing sufficient time for caspase-3 to cleave the substrate. Subsequently, the optical density (OD) of each well was precisely detected at a wavelength of 405 nm using a microplate reader (BioTek, EXL800). The absorbance value obtained is directly proportional to the amount of pNA released, and thus, to the level of active caspase-3. To ensure the statistical reliability and reproducibility of the results, each experiment was conducted in triplicate, and the entire procedure was repeated independently at least three times, providing robust data to confirm the caspase-dependent nature of apoptosis induced by PES.
Western Blotting
Western blotting is a powerful molecular biology technique employed to detect and quantify specific proteins within cell lysates, providing insights into protein expression levels, post-translational modifications like phosphorylation, and protein cleavage events. For this study, cells were initially lysed using RIPA lysis buffer, procured from Beyotime, which was supplemented with 0.5% cocktail protease inhibitor, obtained from Roche. This protease inhibitor cocktail is crucial for preventing the degradation of cellular proteins by endogenous proteases during the lysis process. The resulting cell lysates were then subjected to centrifugation at 12,000 g for 10 minutes at 4 degrees Celsius to separate soluble proteins from insoluble cellular debris, and the protein-rich supernatants were carefully collected. The total protein concentration in each supernatant was then meticulously measured using the bicinchoninic acid (BCA) method, with bovine serum albumin (BSA) serving as a standard curve for accurate quantification.
To prepare for electrophoresis, equal amounts of denatured proteins from each sample were mixed with a 5X loading buffer and then loaded onto 10% SDS-PAGE (sodium dodecyl sulfate–polyacrylamide gel electrophoresis) gels. SDS-PAGE separates proteins primarily based on their molecular weight. Following electrophoretic separation, the separated proteins were transferred from the gel onto polyvinylidene difluoride (PVDF) membranes, acquired from Bio-Rad, Hercules, California, USA. PVDF membranes are chosen for their excellent protein binding capacity. After the transfer, the membranes were blocked with a solution of 5% non-fat milk in TBST (Tris-buffered saline with Tween 20) for 1 hour at room temperature. This blocking step is crucial to prevent non-specific binding of antibodies in subsequent steps. Following blocking, the membranes were incubated with desired primary antibodies overnight at 4 degrees Celsius. These primary antibodies are highly specific, binding only to their target proteins. After three thorough washes (5 minutes each) in TBST to remove unbound primary antibodies, the membranes were then incubated with corresponding horseradish-peroxidase (HRP)-conjugated secondary antibodies for 1 hour at room temperature. These secondary antibodies bind to the primary antibodies and carry the HRP enzyme. Finally, protein expression and phosphorylation were detected using enhanced chemiluminescence (ECL) systems, also from Bio-Rad. The HRP enzyme catalyzes a reaction that produces light, which can then be detected and quantified, providing a visual representation and quantitative measure of antibody-bound proteins.
A comprehensive panel of primary antibodies was utilized in this study to probe various cellular pathways. GAPDH (glyceraldehyde-3-phosphate dehydrogenase), obtained from Proteintech (Peking, China), served as a ubiquitous housekeeping protein and was used as a loading control to ensure equal protein loading across all lanes. Antibodies against Vimentin and E-cadherin (from Santa Cruz Biotechnology) were used to assess epithelial-mesenchymal transition (EMT) markers. Antibodies for cleaved caspase-3, cleaved caspase-9 (from Cell Signaling Technology), and cleaved PARP (from Santa Cruz Biotechnology) were employed to detect activated apoptotic executioners and their substrates, confirming programmed cell death. Hsp70 (from Cell Signaling Technology) was used to monitor the expression of the target protein. Antibodies for Akt, p-Akt (phosphorylated Akt), ERK, and p-ERK (phosphorylated ERK) (all from Cell Signaling Technology) were used to assess the activation status of key cell survival and proliferation signaling pathways. Antibodies against p21, cyclin A, and CDK2 (all from Santa Cruz Biotechnology) were used to investigate cell cycle regulatory proteins. MMP9 (from Santa Cruz Biotechnology) was used to assess matrix metalloproteinase activity related to invasion. Finally, antibodies against death receptor 4 (DR4) and death receptor 5 (DR5) (from Santa Cruz Biotechnology) were used to evaluate components of the extrinsic apoptotic pathway. The secondary antibodies utilized were horseradish-peroxidase-conjugated anti-mouse IgG or anti-rabbit IgG, both obtained from Kerui Tech, Wuhan, China, selected based on the species origin of the primary antibodies.
In Vivo Xenograft Model
To evaluate the antitumor efficacy of PES in a physiologically relevant context and to bridge the gap between *in vitro* findings and potential clinical applications, an *in vivo* xenograft model of lung cancer was established. Athymic female nude mice, at 6 weeks of age, were specifically chosen for this model due to their immunodeficient status, which allows for the successful engraftment and growth of human cancer cells without immune rejection. These mice were procured from Beijing HFK Bioscience Co. Ltd. (Beijing, China) and housed under strict pathogen-free conditions to ensure animal health and minimize experimental variability. All procedures involving animal care and use were meticulously reviewed and approved by the Medical Ethics Committee of Wuhan University, ensuring adherence to ethical guidelines and animal welfare regulations.
To establish the tumor xenografts, A549 human NSCLC cells were utilized. A suspension of 1 x 10^7 A549 cells was prepared in Matrigel, a solubilized basement membrane matrix obtained from BD Biosciences, which promotes tumor growth and engraftment *in vivo*. This cell-Matrigel mixture was then inoculated subcutaneously into the flanks of the nude mice, allowing for the formation of palpable tumors. Once the tumors reached a palpable size of approximately 5 x 5 mm^2, signifying successful engraftment and initial tumor growth, twelve mice bearing evident tumors were arbitrarily and randomly assigned to two experimental groups: a PBS control group and a PES treatment group, with each group comprising six mice. This randomization helps to minimize bias in the study. The treatment regimen involved administering either a single intraperitoneal injection of PES at a dose of 20 mg/kg body weight or an equivalent volume of PBS (phosphate-buffered saline) as a vehicle control. Injections were administered every two days to maintain consistent drug exposure. The treatment period extended for 3 weeks, allowing sufficient time to assess the long-term impact of PES on tumor progression. Following the 3-week treatment duration, the mice were humanely euthanized using carbon dioxide, a widely accepted method.
Upon euthanasia, the therapeutic efficacy of PES was meticulously evaluated by measuring several critical parameters related to tumor burden. These included monitoring changes in the mice’s body weight, directly measuring the weight of the excised tumors, and calculating the tumor volume. Tumor volume was precisely determined using the standard formula: 0.5 multiplied by the length of the tumor multiplied by the square of its width (0.5 x length x width^2). After these measurements, tumor samples were meticulously collected and fixed in 10% neutral buffered formalin, a common fixative that preserves tissue architecture. For detailed histological analysis, hematoxylin and eosin (H&E) staining, a routine method for visualizing tissue morphology, and immunohistochemistry, a technique for detecting specific proteins within tissue sections, were performed on the collected tumor samples, following previously established protocols. These *in vivo* studies provided crucial evidence regarding the antitumor activity of PES in a preclinical setting.
Statistical Analysis
To ensure the scientific rigor and validity of the experimental findings, all quantitative data obtained from the various assays were consistently expressed as the mean ± standard deviation (mean ± SD). This statistical representation provides a measure of the central tendency and the dispersion or variability of the data points around that mean. Statistical analysis was meticulously performed using SPSS software, specifically version 19.0, obtained from SPSS, Chicago, Illinois, USA, a widely recognized and robust statistical package. The significance of the difference between any two experimental groups was determined using the Student’s t-test, an appropriate statistical test for comparing the means of two independent samples. For all analyses, a p-value of less than 0.05 (P < 0.05) was considered to be statistically significant. This widely accepted threshold indicates that there is less than a 5% probability that the observed difference between groups occurred purely by random chance, thereby lending confidence to the conclusion that the difference is a genuine effect of the experimental manipulation.
Results
PES Reduces Cell Viability of Human NSCLC Cells
To systematically determine the impact of 2-phenylethyenesulfonamide (PES) on the viability and proliferation of human non-small cell lung cancer (NSCLC) cells, two representative cell lines, A549 and H460, were exposed to varying concentrations of the compound for different durations. Following treatment, cell viability was quantitatively assessed using the Cell Counting Kit-8 (CCK-8) assay. The experimental design involved treating A549 and H460 cells with a series of increasing PES concentrations, specifically ranging from 2.5 to 40 micromolar, and evaluating their viability after both 24 and 48 hours of exposure.
The results from the CCK-8 assays demonstrated a clear and compelling pattern: PES induced a significant loss of viability in A549 cells in a manner that was both dose-dependent and time-dependent. This means that as the concentration of PES increased, and as the duration of exposure lengthened, a greater reduction in cell viability was observed. For A549 cells, the half-maximal inhibitory concentration (IC50), which represents the concentration of PES required to inhibit 50% of cell viability, was calculated to be 44.9 micromolar after a 24-hour treatment period. This potency was further enhanced with prolonged exposure, as the IC50 value decreased to 25.7 micromolar after a 48-hour PES treatment. Similar robust and consistent results were observed in the H460 cell line. For H460 cells, the IC50 values were determined to be 40.1 micromolar after 24 hours of PES treatment, and this efficacy improved to 24.3 micromolar after 48 hours of exposure. These quantitative findings collectively provide strong evidence that PES is an efficient inhibitor of cell proliferation in both A549 and H460 human NSCLC cell lines, highlighting its direct cytotoxic or cytostatic effects on these cancer cells.
PES Suppresses the Migration of Human NSCLC Cells
Accumulating scientific evidence has unequivocally demonstrated that the metastatic activity of cancer cells constitutes a critically important and devastating mechanism contributing to cancer mortality and invasive progression. The ability of cancer cells to migrate from their primary tumor site and colonize distant organs is a key driver in the development and advancement of malignant disease. In order to thoroughly investigate the impact of 2-phenylethyenesulfonamide (PES) on the migratory capabilities of human non-small cell lung cancer (NSCLC) cells, two distinct and complementary *in vitro* assays were employed: the wound healing assay and the Transwell migration assay.
In the wound healing assay, which assesses collective cell migration, the experimental results clearly indicated that, when compared to untreated control groups, PES significantly suppressed the process of wound healing in both A549 and H460 cells. This reduced ability of the cells to migrate into the created cell-free gap provided preliminary evidence of an anti-migratory effect. To further validate this observation and crucially, to exclude any potential confounding effects that reduced cell proliferation might have on the apparent reduction in migration, the Transwell migration assay was subsequently utilized. This assay provides a more direct and quantitative measure of individual cell migration through a porous membrane. Consistent with the wound healing assay results, the inhibitory effect of PES on cell migration was definitively confirmed by the Transwell migration assay. In this assay, a significantly reduced number of migrated A549 and H460 cells were observed after treatment with PES for 48 hours, indicating a profound suppression of their invasive potential. These consistent findings from both assays collectively and strongly suggest that PES possesses the capacity to effectively inhibit the migratory behavior of human NSCLC cells, implying a potential therapeutic benefit in impeding the metastatic spread of lung cancer.
PES Induces Cell Cycle Arrest in Human NSCLC Cells
To systematically unravel the precise molecular mechanism by which 2-phenylethyenesulfonamide (PES) exerts its inhibitory effects on the proliferation of human non-small cell lung cancer (NSCLC) cells, flow cytometry was utilized to conduct a comprehensive analysis of the cell cycle distribution in A549 and H460 cell lines. These cells were treated with a specific concentration of PES (20 micromolar) for a duration of 24 hours, after which their DNA content was stained to allow for cell cycle phase quantification.
The flow cytometry results provided clear and compelling evidence that PES treatment markedly interfered with the normal progression of the cell cycle in both A549 and H460 cells. This disruption was characterized by a significant and distinct decrease in the percentage of cells residing in the S phase, which is the crucial stage of DNA synthesis, indicating an impediment to DNA replication. Concomitantly, a notable increase was observed in the percentage of cells accumulating in the G0/G1 phase, the quiescent or initial growth phase of the cell cycle. This accumulation strongly suggested that PES induced a G0/G1 phase cell cycle arrest, effectively preventing cells from advancing into DNA synthesis and subsequent division. More specifically, in A549 cells, treatment with PES led to a substantial increase in the fraction of cells in G0/G1 phase, rising from a baseline of 32.8% in control cells to a pronounced 54.1%. Correspondingly, the proportion of cells in the S phase dramatically decreased from 55.4% in control cells to 32.8% following PES treatment. A remarkably similar pattern was observed in H460 cells, where PES treatment caused an increase in the G0/G1 phase fraction from 34.2% to 48.4%, while simultaneously reducing the S phase fraction from 44.6% to 33.5%. It is particularly noteworthy that throughout these analyses, no marked or statistically significant change was detected in the percentage of cells residing in the G2/M phase (the preparatory and mitotic phases of the cell cycle) in either A549 or H460 cells, further specifying the G0/G1 checkpoint as the primary site of PES-induced arrest.
These findings from flow cytometry were further corroborated and molecularly validated by Western blotting analyses, which provided insights into the expression levels of key cell cycle regulatory proteins. The Western blotting results consistently indicated that PES treatment effectively inhibited the expression of both cyclin A and CDK2 (Cyclin-Dependent Kinase 2). These proteins are well-established positive regulators and crucial markers of cell cycle progression, and their downregulation directly contributes to cell cycle arrest. Conversely, the expression of p21, a cyclin-dependent kinase inhibitor (CDKI) and a potent negative regulator of cell cycle progression, was found to be notably increased following PES treatment. The upregulation of p21 is consistent with a G0/G1 phase arrest, as p21 typically halts cell cycle progression by inhibiting the activity of cyclin-CDK complexes.
Beyond its impact on cell proliferation, the study also explored how PES influenced cell migration and invasion. Given that inhibition of cell migration can, in some contexts, be partly attributed to or confounded by an inhibition of cell proliferation, Western blotting was additionally utilized to detect the expression of genes specifically associated with cell invasion and metastatic potential. Consistent with the functional observations from the wound healing assay, the Western blotting results provided molecular evidence that PES treatment actively increased the expression of E-cadherin, a critical epithelial marker involved in cell-to-cell adhesion, and simultaneously reduced the expression of vimentin and MMP9 (Matrix Metalloproteinase-9). Vimentin is a mesenchymal marker, and its downregulation, along with the upregulation of E-cadherin, is indicative of a reversal or inhibition of the epithelial-mesenchymal transition (EMT), a cellular process frequently associated with enhanced cancer cell migration and invasion. Similarly, MMP9 is an enzyme that degrades components of the extracellular matrix, facilitating cell invasion, and its reduction signifies decreased invasive potential. Therefore, these molecular findings strongly suggest that PES inhibited human NSCLC cell migration and invasion, at least in part, via its intricate regulation of the epithelial to mesenchymal transition process, providing a multifaceted mechanism for its antitumor activity.
PES Induces Cell Apoptosis in Human NSCLC Cells
To comprehensively determine whether the observed inhibitory effect of 2-phenylethyenesulfonamide (PES) on cell viability was intrinsically linked to the induction of programmed cell death, specifically apoptosis, a series of detailed experiments were conducted. A549 and H460 human non-small cell lung cancer (NSCLC) cells were treated with or without PES (at a concentration of 20 micromolar) for a duration of 24 hours, and the extent of apoptosis was then meticulously analyzed using flow cytometry.
The flow cytometry results provided clear and compelling evidence that PES treatment indeed triggered a significant induction of apoptosis in both cell lines. This was unequivocally validated by a marked increase in the percentage of Annexin V-positive cells, a hallmark indicator of early apoptosis where phosphatidylserine translocates to the outer cell membrane. Quantitative analysis of the Annexin V-positive cell populations further solidified these findings, demonstrating a statistically significant increase in apoptotic cells following PES treatment compared to untreated controls.
In addition to the Annexin V-FITC assay, Western blot analysis was employed to provide further molecular confirmation of PES’s pro-apoptotic effects. A549 and H460 cells were treated with or without PES (at concentrations of 10 or 20 micromolar) for 48 hours. Whole-cell extracts were then harvested, and the protein content was subjected to Western blotting to detect the activation of key apoptotic executioners. The Western blot results demonstrably showed that PES treatment led to a significant increase in the expression of cleaved caspase-3, cleaved caspase-9, and cleaved PARP (Poly (ADP-ribose) polymerase) in both A549 and H460 cells. The cleavage of pro-caspase-9 signifies its activation as an initiator caspase, typically associated with the intrinsic mitochondrial apoptotic pathway. Activated caspase-9 then cleaves and activates downstream effector caspases, such as caspase-3. The subsequent cleavage of PARP by active caspase-3 is a widely recognized biochemical hallmark of apoptosis, indicating the irreversible commitment to programmed cell death.
To further quantify the functional activation of caspases, specifically caspase-3, an enzyme activity assay was performed. The results from the caspase-3 activity assay confirmed the Western blot findings, indicating that the enzymatic activity of caspase-3 was dose-dependently increased by PES treatment in both A549 and H460 cells. This dose-dependent activation further underscored the direct role of PES in triggering the caspase cascade. Taken together, these comprehensive results strongly suggested that PES induces apoptosis in human NSCLC cells, at least in part, through a caspase-dependent intrinsic mitochondrial pathway, thereby effectively promoting programmed cell death as a mechanism for its antitumor activity.
PES Inhibits Activation of Akt and ERK Pathway in Human NSCLC Cells
The AKT and MAPK (Mitogen-Activated Protein Kinase) signaling pathways are widely recognized as indispensable components in the complex regulatory networks governing cell survival, proliferation, and the execution of apoptosis. Their aberrant activation is frequently observed in various cancers, contributing to tumor aggressiveness and resistance to therapy. Previous scientific reports have consistently highlighted that AKT and ERK (Extracellular signal-Regulated Kinase), a key component of the MAPK pathway, are central modulators of cell proliferation. Moreover, the activation of both AKT and ERK, typically through phosphorylation, has been directly correlated with increased tumor aggressiveness and a poorer prognosis in non-small cell lung cancer (NSCLC) patients.
To precisely determine the impact of 2-phenylethyenesulfonamide (PES) on these pivotal AKT and ERK signaling pathways, A549 and H460 cells were treated with either a vehicle control or varying concentrations of PES (10 or 20 micromolar) for a duration of 48 hours. Whole-cell extracts were then prepared and subjected to Western blotting analysis to assess the phosphorylation status of AKT and ERK, which serves as an indicator of their activation. The results unequivocally demonstrated that PES treatment led to a dose-dependent decrease in the levels of phosphorylated AKT (p-AKT) and phosphorylated ERK (p-ERK) in both A549 and H460 cells. This direct inhibition of AKT and ERK phosphorylation indicates that PES effectively dampens these crucial pro-survival and pro-proliferative signaling cascades, contributing significantly to its observed antiproliferative and pro-apoptotic effects.
In addition to suppressing these survival pathways, Western blotting analysis also revealed another important molecular alteration: the expression levels of death receptor 4 (DR4) and death receptor 5 (DR5) were found to be increased following PES treatment. DR4 and DR5 are key transmembrane receptors that mediate the extrinsic apoptotic pathway, and their upregulation suggests that PES sensitizes cancer cells to external apoptotic stimuli.
Previous studies have consistently indicated that Hsp70 is fundamentally essential for NSCLC growth regulation, with its overexpression being a common feature in NSCLC cells compared to their normal counterparts. Given that PES is characterized as a specific Hsp70 inhibitor, the study also meticulously evaluated the direct effect of PES on Hsp70 protein levels in human NSCLC cells. Surprisingly, despite its classification as an Hsp70 inhibitor, Western blotting results showed that PES treatment did not lead to a reduction in the overall expression levels of Hsp70 protein in either A549 or H460 cells. This particular finding suggests that PES might not function by decreasing the *amount* of Hsp70 protein but rather by inhibiting its functional activity. This proposed mechanism aligns with the understanding that PES is known to specifically interact with and disrupt the ATPase binding domain of Hsp70, thereby impairing its crucial protein chaperoning activity without necessarily altering its total protein levels.
PES-Induced Cell Proliferation Inhibition is Involved in the Regulation of Hsp70 in Human NSCLC Cells
To further unequivocally validate that the observed decrease in cell viability induced by 2-phenylethyenesulfonamide (PES) treatment was indeed specifically caused by its action on Hsp70, a series of critical experiments involving genetic manipulation of Hsp70 expression were performed in A549 cells.
Firstly, to assess the effect of Hsp70 overexpression, A549 cells were transiently transfected with either a plasmid designed to overexpress Hsp70 (pSG5-Hsp70) or a vehicle control plasmid (pSG5). After a 4-hour transfection period, these cells were then treated with or without PES (20 micromolar) for an additional 44 hours. Western blotting confirmed that Hsp70 was successfully overexpressed in the designated A549 cells. Subsequently, cell viability was meticulously assessed using the CCK-8 assay. The results demonstrated that while overexpression of Hsp70 by itself did not significantly increase cell viability, it effectively and notably attenuated the inhibitory efficiency of PES on cell proliferation. This crucial finding suggests that higher intracellular levels of functional Hsp70 can partially counteract the antiproliferative effects of PES, providing strong correlational evidence for Hsp70 as a target.
Secondly, to complement the overexpression studies and provide further direct evidence of Hsp70′s role, Hsp70 expression was specifically knocked down in A549 cells using small interfering RNA (siRNA) designed to target Hsp70 mRNA (siHsp70). A non-targeting control siRNA (siControl) was used for comparison. Western blotting analyses confirmed a significant reduction in Hsp70 protein expression following transfection with siHsp70. Furthermore, and importantly, Hsp70 knockdown by siHsp70 alone resulted in a substantial decrease in cell viability, specifically by 58.8%, indicating that Hsp70 is indeed critical for the basal survival and proliferation of these NSCLC cells. Most critically, A549 cells in which Hsp70 was knocked down by siHsp70 exhibited significantly enhanced sensitivity to PES-induced cell proliferation inhibition. This means that with reduced Hsp70 levels, the cells were more susceptible to the growth-inhibitory effects of PES. Taken collectively, these compelling reciprocal results, where both overexpression and knockdown of Hsp70 directly modulated the efficacy of PES, unequivocally suggest that PES inhibits the proliferation of human NSCLC cells specifically through an Hsp70-dependent mechanism, solidifying Hsp70 as its primary therapeutic target.
PES Sensitizes NSCLC Cell Lines to TRAIL-Induced Cell Proliferation Inhibition and Apoptosis
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), also known as Apo-2L, has emerged as a highly attractive antitumor therapeutic agent due to its remarkable ability to selectively trigger apoptosis in a broad spectrum of tumor cells while exhibiting minimal or no toxicity to most normal, healthy cells. This selective cytotoxicity stems from TRAIL’s mechanism of action: it can specifically induce apoptosis in tumor cells by recognizing and binding to cognate death receptors, namely death receptor 4 (DR4) and death receptor 5 (DR5), which are typically expressed on the surface of cancer cells. Given our earlier observation that PES treatment leads to an upregulation of both DR4 and DR5 expression, we hypothesized that combining PES with TRAIL might lead to a synergistic enhancement of cell proliferation inhibition and apoptosis induction in NSCLC cells.
To test this hypothesis, A549 and H460 cells were subjected to various treatment conditions: either PES (20 micromolar) alone, TRAIL (40 ng/ml) alone, or a combination of both PES and TRAIL, for a period of 24 hours. Following treatment, the cells were harvested and meticulously analyzed for cell viability using the CCK-8 assay, for apoptosis induction using flow cytometry (Annexin V-FITC/PI staining), and for caspase-3 activation using a specific enzyme activity assay.
The results from the cell viability assays provided compelling evidence of synergistic interaction. In A549 cells, treatment with PES alone reduced cell viability by 67.2%, and TRAIL alone reduced it by 84.9%. However, when PES and TRAIL were co-administered, the cell viability was dramatically inhibited to a mere 28.5%, indicating a potent synergistic effect beyond simple additive reduction. Similarly, in H460 cells, PES alone reduced viability by 59.9%, and TRAIL alone by 66.1%, but co-treatment resulted in a significantly lower viability of 19.7%. Flow cytometry results further supported this synergy, suggesting that co-treatment with PES and TRAIL led to a greater degree of cell apoptosis compared to PES treatment alone. Interestingly, TRAIL treatment alone, at the concentration used, did not result in a marked induction of apoptosis in either A549 or H460 cells, indicating an inherent resistance to TRAIL in these NSCLC cell lines. Quantitative analysis of Annexin V-positive cells clearly illustrated the enhanced apoptotic effect of the combination therapy.
As expected for an enhanced apoptotic effect, the co-treatment of A549 and H460 cells with PES and TRAIL resulted in a marked and synergistic increase in caspase-3 activation, when compared to either PES or TRAIL treatment alone. This heightened caspase-3 activity signified a more robust triggering of the downstream apoptotic cascade. Furthermore, Western blotting analysis for cleaved PARP, a direct substrate of active caspase-3, corroborated these findings. Treatment with TRAIL alone did not induce detectable cleavage of the PARP protein, aligning with the observed TRAIL resistance. However, co-treatment of cells with PES and TRAIL led to a significantly enhanced expression of cleaved PARP compared to PES treatment alone. Taken together, these comprehensive results strongly suggested that PES effectively sensitizes A549 and H460 cells to TRAIL-induced cell proliferation inhibition and apoptosis, presenting a promising strategy for combination therapy to overcome intrinsic TRAIL resistance in NSCLC.
In Vivo Effects of PES on A549 Lung Tumor Xenografts
To comprehensively evaluate the *in vivo* antitumor activity of 2-phenylethyenesulfonamide (PES) in a preclinical model, a mouse xenograft model of lung cancer was established using human A549 cells. Athymic female nude mice were inoculated subcutaneously into their flanks with 1 x 10^7 A549 cells, suspended in Matrigel, to facilitate tumor engraftment and growth. After 7 days, once the mice developed visibly evident tumors with an approximate size of 5 x 5 mm^2, they were randomly assigned to either a PBS control group or a PES treatment group, with six mice in each group to ensure statistical validity.
The treatment regimen involved administering either PES at a dose of 10 mg/kg body weight or an equivalent volume of phosphate-buffered saline (PBS) as a vehicle control, via intraperitoneal injection every two days. This schedule ensured consistent drug exposure throughout the treatment period. After a 3-week treatment duration, the mice were humanely euthanized, and their tumors were meticulously measured and weighed to assess the therapeutic efficacy. The results unequivocally demonstrated that, when compared to the PBS control group, PES treatment led to a significant reduction in both tumor volume and tumor weight, providing robust evidence of its *in vivo* antitumor activity. Moreover, a critical observation regarding safety was made: no significant loss of mouse body weight was observed in the PES treatment groups, which is a common indicator of systemic toxicity associated with anticancer agents. This suggests that the dose of PES used was well-tolerated by the animals.
To further assess potential PES-associated toxicity in the mice and to gain deeper insights into its mechanism of action within the tumor, major organs (liver, kidney, spleen, and lung) and tumor samples were carefully dissected. These tissues were then subjected to standard hematoxylin and eosin (H&E) staining for general histological analysis and immunohistochemistry analysis for specific molecular markers. The H&E staining results indicated no notable histopathological differences or signs of adverse systemic toxicity between the PES-treated and PBS control groups across the examined major organs, confirming the compound’s safety profile at the tested dosage *in vivo*. In addition, immunohistochemistry analysis of the tumor tissues obtained from the PES-treated mice further substantiated the observed growth inhibition and provided mechanistic insights. These tumor tissues exhibited clear signs of increased apoptosis, as evidenced by positive staining for cleaved caspase-3 and TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling), both widely recognized markers of programmed cell death. Concomitantly, a reduction in cell proliferation was indicated by decreased staining for Ki-67, a marker of cellular proliferation. Taken collectively, these comprehensive *in vivo* data strongly suggest that PES exerts a potent inhibitory effect on lung carcinoma xenograft growth by directly inducing tumor cell apoptosis in mice. Furthermore, the absence of marked signs of systemic toxicity in the treated mice indicated that the dosage of PES utilized in this study was well-tolerated and safe, underscoring its promising therapeutic window for NSCLC treatment.
Discussion
Over the past several decades, lung cancer has regrettably emerged as one of the most frequently diagnosed malignancies, and its associated morbidity and mortality rates have shown a marked and concerning increase across the globe. This escalating burden underscores the urgent and imperative need for the discovery and development of more effective therapeutic agents and innovative strategies for the comprehensive treatment of lung cancer. Cancer, in its fundamental nature, is widely recognized as a highly complex disease characterized by the aberrant upregulation and dysregulation of multiple oncogenic proteins. These proteins often play pivotal roles in activating various pro-survival signaling pathways within the cancerous cells, thereby promoting their uncontrolled growth and resistance to death. The chaperone function of the heat shock protein (Hsp) family, a group of molecular chaperones, plays a critical role in maintaining the stability and function of many of these oncogenic proteins. Consequently, accumulating scientific results have convincingly indicated that targeting the molecular chaperone function of Hsp family members represents a highly attractive and promising therapeutic avenue for the effective treatment of cancer.
In this comprehensive study, the multifaceted biological effects of 2-phenylethyenesulfonamide (PES), a small molecular inhibitor specifically targeting Hsp70, were meticulously determined in human non-small cell lung cancer (NSCLC) cells, both under controlled *in vitro* laboratory conditions and within a relevant *in vivo* animal model. Firstly, our *in vitro* investigations revealed that PES exhibited a robust and consistent ability to inhibit cell proliferation in human NSCLC cells in a manner that was both time-dependent and dose-dependent. Complementing these proliferation findings, results from the wound healing assay demonstrated that, when compared to vehicle-treated control cells, the wound width in PES-treated cells was significantly wider, indicating that PES could efficiently inhibit the migratory capabilities of human NSCLC cells. While acknowledging that inhibition of cell migration might, in some instances, be partly attributable to or confounded by the concomitant inhibition of cell proliferation, the results from the more quantitative Transwell migration assay provided definitive confirmation. This assay demonstrated a significant reduction in the number of migrated A549 and H460 cells after 48 hours of PES treatment, thus unequivocally confirming the direct inhibitory effect of PES on cell migration. Epithelial-mesenchymal transition (EMT) is a crucial cellular phenomenon widely recognized for its pivotal role in regulating tumor progression, particularly metastasis, and its inhibition is considered a potent target for innovative cancer therapies. To investigate if PES affected EMT, we analyzed the expression of key molecular markers associated with cell invasion and EMT. Our Western blotting results demonstrated that PES reduced the expression of MMP-9 and Vimentin, both of which are indicators of increased invasiveness and mesenchymal phenotype, while simultaneously increasing the expression of the epithelial marker, E-cadherin, which is associated with cell-to-cell adhesion and a less invasive phenotype. Therefore, these molecular findings strongly suggest that PES inhibited human NSCLC cell migration and invasion, at least in part, via its intricate regulation of the EMT process, providing a multifaceted mechanism for its antitumor activity.
In addition to its inhibitory effect on cell growth and migration, PES was found to induce cell cycle arrest and apoptosis, both crucial mechanisms for anticancer agents. Flow cytometry results unequivocally suggested that PES disrupted the normal progression of the cell cycle, which was clearly evidenced by a markedly increased percentage of NSCLC cells accumulating in the G0/G1 phase. Furthermore, supporting these findings at a molecular level, cell cycle progression markers such as CDK2 and Cyclin A were observed to be decreased following PES treatments, while the expression of the cyclin-dependent kinase inhibitor, p21, a negative regulator of cell cycle progression, was increased. The precise mechanism by which PES induces cell death, whether through apoptotic or non-apoptotic pathways, has been a subject of some controversy in previous studies. For instance, PES was reported to induce cell death in primary effusion lymphoma through the induction of lysosomal cathepsin D release, a mechanism distinct from canonical apoptotic processes. Conversely, other reports indicated that PES induced apoptotic cell death in pancreatic cell lines via both caspase-dependent and -independent pathways. Our results, however, clearly demonstrated that PES specifically induced cell apoptosis in human NSCLC cells through a caspase-dependent mechanism. Caspase activation represents the central and key event of apoptosis. As anticipated, following treatment of human NSCLC cells with PES, the expression of cleaved caspase-9, an initiator caspase, was increased. This activation was accompanied by a concomitant increase in cleaved caspase-3, a key executioner caspase, and cleaved PARP protein, a well-known substrate of active caspases. Furthermore, the results from the direct caspase-3 activation assay corroborated these findings, quantitatively confirming the elevated enzymatic activity of caspase-3. These collective results strongly indicate that PES induces cell apoptosis in human NSCLC cells, at least in part, in a caspase-dependent manner, highlighting its ability to trigger programmed cell death.
Beyond its direct effects on cell cycle and apoptosis, multiple signaling pathways, particularly the ERK and AKT signal transduction pathways, are recognized for their pivotal roles in governing cell survival, proliferation, and the regulation of apoptosis. Our results consistently demonstrated that PES decreased the level of phosphorylated (activated) AKT and ERK in human NSCLC cells. These findings are in strong agreement with previous reports concerning lung cancer cells treated with other agents, such as VER-155008, further validating the inhibitory effect of Hsp70 targeting on these pathways. In addition to its role in protecting cells from repeated exposure to harmful stimuli, Hsp70 actively assists in the proper folding of nascent polypeptides and proteins, underscoring the critical importance of Hsp70 in the growth and survival of tumor cells, which often rely heavily on chaperone machinery to manage misfolded oncoproteins. Prior reports have indicated that the expression of Hsp70 was inhibited by compounds like triptolide, leading to pancreatic cancer cell death. Similarly, quercetin and gemcitabine synergistically enhanced apoptosis in lung cancer cells via inhibition of Hsp70, highlighting its potential as a combination therapy target. Given that PES has been specifically identified as an Hsp70 inhibitor, its precise regulatory effect on Hsp70 was meticulously evaluated in human NSCLC cells. Although the overall expression level of Hsp70 protein was not detectably reduced by PES, our experiments involving Hsp70 overexpression provided crucial insights: overexpression of Hsp70 significantly attenuated the inhibitory effect of PES on cell proliferation in A549 cells. This suggests that PES primarily targets the ATPase binding domain of Hsp70, thereby inhibiting its crucial protein chaperoning activity without necessarily diminishing its total protein quantity. As expected, and further confirming the specific role of Hsp70, the targeted knockdown of Hsp70 using siRNA in A549 cells significantly enhanced the sensitivity of these cells to PES-induced cell growth inhibition, providing compelling genetic evidence for the Hsp70-dependent mechanism of action of PES.
TRAIL, also referred to as Apo-2L, is considered an attractive antitumor therapeutic agent due to its ability to selectively induce apoptosis in tumor cells by binding to death receptor 4 (DR4) and death receptor 5 (DR5) on the cell surface, while sparing most normal cells. Given our observation that PES treatment led to an increase in the expression of DR4 and DR5, we investigated the potential synergistic effects of combining PES with TRAIL on human NSCLC cells. Our results clearly indicated that combination treatment with PES and TRAIL synergistically inhibited cell proliferation and markedly increased the percentage of Annexin V-positive (apoptotic) cells in human NSCLC cells. Interestingly, TRAIL treatment alone in human NSCLC cells did not induce significant apoptosis, suggesting that human NSCLC cells exhibit intrinsic resistance to TRAIL. This intrinsic TRAIL resistance is a common challenge in cancer therapy. Our findings suggest that the mechanisms underlying TRAIL resistance may involve the regulation of death receptors, as the increased expression of DR4 and DR5 following PES treatment effectively sensitized NSCLC cell lines to the pro-apoptotic effects of TRAIL. Even so, the precise and comprehensive mechanisms contributing to TRAIL resistance in human NSCLC cells warrant further dedicated investigation.
In addition to the extensive *in vitro* studies on the effects of PES on human NSCLC cells, this study also crucially investigated the therapeutic efficacy of PES against NSCLC *in vivo* utilizing a mouse xenograft model. Our results unequivocally demonstrated that PES significantly repressed the growth of established tumor xenografts, providing compelling evidence of its antitumor activity in a living organism. This inhibition was quantitatively confirmed by a notable decrease in both the volume and weight of the solid tumors in PES-treated mice compared to controls. Furthermore, a crucial aspect of therapeutic development is safety. Our *in vivo* data indicated that no notable differences were observed among mouse body weight and the histology of major organs (liver, kidney, spleen, and lung) in the PES treatment group compared to the control group. This suggests that PES, when administered at the dosage used in this study, caused few systemic side effects, indicating a favorable safety profile. Histological and immunohistochemical analyses of the tumor tissues from PES-treated mice further revealed that the growth inhibition was associated with increased tumor cell apoptosis, as evidenced by positive staining for cleaved caspase-3 and TUNEL, along with reduced proliferation, indicated by Ki-67 staining. Collectively, these comprehensive data strongly suggest that PES exerts a potent *in vivo* inhibitory effect on lung carcinoma xenograft growth by inducing tumor cell apoptosis in mice. Moreover, the lack of marked signs of systemic toxicity observed with PES in this study indicates that the dosage used *in vivo* was safe and well-tolerated.
In conclusion, the results of this study provide compelling evidence regarding the capabilities of PES, both *in vitro* and *in vivo*, to effectively inhibit cell viability in human non-small cell lung cancer cells. Furthermore, our findings clearly indicate that the mechanism of cell viability inhibition by PES is directly related to its regulation of Hsp70 in human NSCLC cells, validating its role as a targeted Hsp70 inhibitor. Although human NSCLC cells demonstrated intrinsic resistance to TRAIL treatment when administered alone, TRAIL exhibited a potent synergistic effect on cell proliferation inhibition when combined with PES, suggesting a valuable combination therapy strategy. Taken together, these multifaceted findings position PES as a highly promising compound for the future management of human NSCLC. However, the precise mechanisms underlying the synergistic anticancer action of PES and TRAIL warrant further detailed investigation to optimize their combined therapeutic potential.