2-phenylacetylsulfonamide promotes apoptosis of hepatocellular carcinoma by inhibiting HSP70 through PI3K/AKT signaling pathway

2-phenylacetylsulfonamide promotes apoptosis of hepatocellular carcinoma by inhibiting HSP70 through PI3K/AKT signaling pathway | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article 2-phenylacetylsulfonamide promotes apoptosis of hepatocellular carcinoma by inhibiting HSP70 through PI3K/AKT signaling pathway Shengfeng Fu, Haifeng Li, Xiaoqian Zhao, Yu Zhang, Jiali Chen, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8501296/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 5 You are reading this latest preprint version Abstract Heat shock proteins (HSPs) are found widely across various organisms and play a crucial role in tumor and malignancy formation. Among all HSPs, HSP70 is the most extensively researched. 2-phenylacetylsulfonamide (PES) is a covalent inhibitor of HSP70, which has a significant effect on inhibiting wide range of cancer cells. Nevertheless, the effects of PES on hepatocellular carcinoma (HCC) cells are still largely unknown. The objective of this research was to investigate the relationship between PES and apoptosis-related proteins in human HCC, and to assess the potential mechanism of drug action on HCC. This study demonstrated the anti-cancer activity of PES against HCC cells. Cell viability was assessed utilizing the CCK-8 kit, which revealed that PES suppressed the growth of HepG2 cells in a dose-and time-dependent. Furthermore, the data from Hoechst 33342 staining indicate a gradual suppression of HCC cell proliferation with increasing PES concentration. The expression of the target gene HSP70 and various apoptotic genes was quantified using Western blot analysis. The findings demonstrate that PES significantly inhibited the expression of HSP70 and promoted the apoptosis of human HepG2 cells. Moreover, the immunofluorescence findings indicated a notable reduction in the expression of the target gene HSP70 in PES-treated cells of HCC, whereas the expression of Caspase-3 was up-regulated. Furthermore, our findings demonstrated that PES induces apoptosis in HepG2 cells by suppressing the PI3K/AKT signaling pathway. These results demonstrated that PES has anti-tumor activity against human HCC and may be an effective drug for HCC treatment. Biological sciences/Cancer Biological sciences/Cell biology Biological sciences/Drug discovery Biological sciences/Molecular biology Health sciences/Oncology Hepatocellular carcinoma HSP70 PES Apoptosis Figures Figure 1 Figure 2 Figure 3 Figure 4 1. Introduction Hepatocellular carcinoma (HCC) is one of the major reasons for cancer-related mortality throughout the world, accounting for about 90% of all primary liver cancer cases [1] . Early diagnosis and treatment of HCC is virtually impossible due to the lack of specific clinical signs and symptoms [2] . Despite significant progress in treating HCC using modern surgical and chemotherapeutic techniques, the 5-year survival rate remains under 18% [3] . As such, new therapeutic agents and effective treatment strategies are urgently required to improve prognosis of HCC patients. In every living organism, heat shock proteins (HSPs) serve as an ancient defensive mechanism, acting as molecular chaperones to promote the appropriate folding and refolding of misfolded proteins, as well as assisting in the breakdown of damaged and aging cells [4,5] . HSP70 is a protein associated with tumorigenicity and possessing various functions, such as performing as a cellular safeguard, and exhibiting anti-apoptotic and immune functions [6] . Increasingly, the expression of HSP70 has been shown to be significantly elevated in human tumor cells and may influence cancer development through a variety of mechanisms. In contrast, inhibiting or knocking down HSP70 can reduce tumor size and even lead to complete tumor regression [7,8] . An attractive strategy for cancer therapy is therefore to target HSP70. 2-phenylacetylsulfonamide (also referred to as pifithrin-µ or PES), a selective inhibitor of HSP70, was initially thought to be an inhibitor of p53 binding to mitochondria [9] . Mechanically, PES reacts covalently with the sulfhydryl group on HSP70, leading to disruption of the binding between HSP70's co-chaperone and the substrate protein, resulting in inhibition of its activity [10,11] . PES-induced cancer cell death is primarily associated with the accumulation of misfolded proteins, disruption of autophagy and impairment of lysosomal function [12,13,14] . In this study, we aimed to look into the connection between PES, HSP70, and apoptosis-related genes in HCC cells. The findings exhibited that PES suppressed the expression of HSP70 and triggered apoptosis. In this process, we focused on determining the expression of apoptotic genes such as Bax, Bcl-2, Caspase-3, and Caspase-9. In addition, we confirmed that PES induces apoptosis through the inhibition of the PI3K/AKT pathway. 2. Materials and methods 2.1 Reagents and antibodies PES and LY294002 were purchased from Med Chem Express (MCE, Shanghai, China). Antibodies for HSP70 (1:1000), Bax (1:1000), Bcl-2 (1:1000), Caspase-3 (1:500) and GAPDH (1:10000) were obtained from Proteintech (Wuhan, China). Antibody for PI3K (1:1000), P-PI3K (1:1000), AKT (1:1000), P-AKT (1:1000), cleaved Caspase-3 (1:1000) and cleaved Caspase-9 (1:1000) were obtained from Cell Signal Technology (Boston, USA). The secondary antibodies were horseradish-peroxidase-conjugated secondary anti-mouse IgG or anti-rabbit IgG (Proteintech, Wuhan, China). 2.2 Cell culture and treatment The HCC cell lines, HepG2, Hep3B, HUH-7 were purchased from Procell (Wuhan, China). Cell culture were performed in DMEM supplemented with 10% Every Green fetal bovine serum (Zhejiang, China) and 1% (v/v) penicillin/ streptomycin and placed in a humidified atmosphere with 5% CO2 at 37 ℃. PES was solubilized with DMSO (Sigma, St. Louis, MO, USA). 2.3 Determination of HSP70 Blood samples were collected from 6 patients with clinically diagnosed hepatocellular carcinoma (experimental group) and 6 normal people (control group). After centrifugation, the upper layer of serum was taken and stored in a refrigerator at -80℃. Six cases of liver cancer tissues and adjacent tissues were collected and stored in a refrigerator at -80℃ under liquid nitrogen. Then the expression of HSP70 in serum and tissue was detected by ELISA kit, immunohistochemistry and Immunofluorescence. The design and reporting of this study comply with the requirements of the Declaration of Helsinki and have been approved by the Clinical Research Ethics Committee of Taizhou People's Hospital. We confirm that all research was conducted in accordance with relevant guidelines and regulations, and informed consent was obtained from all participants and/or their legal guardians. 2.4 Screening of HCC cell lines Three cell lines, HepG2, Hep3B and HUH-7, were selected as test subjects based on their different expression levels of HSP70, and then the content of HSP70 in these three cells was determined. 2.5 Cell viability assay To determine cell viability, the Cell Counting Kit-8 (CCK-8) assay kit was utilized. In brief, HepG2 cells were incubated overnight in 96-well plates, maintained at a cell density of 6×10 3 per 100 µl. After 24 and 48 hours of treatment with different concentrations of PES, 10µl CCK-8 solution was added to each well, followed by incubation at 37 ℃ for a duration of 2 hours. The absorbance was accurately measured using a microplate reader (Biotek) specifically set at a wavelength of 450 nm. 2.6 Edu incorporation assay The capacity of cell proliferation was evaluated through the 5-ethynyl−2-deoxyuridine (EdU) incorporation assay, which was conducted after subjecting the cells to 24 hours of treatment with diverse concentrations of PES, following the manufacturer's instructions (Beyotime, Shanghai, China). The images were observed and recorded using an inverted fluorescence microscope (Leica). 2.7 Caspase−3 activity assay The Caspase−3 Activity Kit (Beyotime Institute of Biotechnology, Guangzhou, China) was utilized to measure Caspase−3 activity, strictly adhering to the manufacturer's protocol. This method is based on the ability of caspase−3 to catalyze the production of yellow pNA from the substrate acetyl-Asp-GluVal-Asp-p-nitroanilide (Ac-DEVD-pNA), and therefore the activity of caspase−3 can be detected by measuring the absorbance. After treatment, cells were harvested and lysed with lysis buffer. Lysates, assay buffer, and caspase−3 substrate were incubated together at a temperature of 37°C for a duration of 2 hours. Subsequently, the absorbance of the samples was accurately measured at a wavelength of 405 nm. 2.8 Immunofluorescence Cells were seeded at a density of 8 × 10 4 cells per well within 12-well plates. After 24 hours of treatment with various concentrations of PES, the cells were immobilized with a solution containing 4% paraformaldehyde. The cells were incubated with HSP70 (1:200) and Caspase−3 (1:500) overnight at 4℃ after blocking with immunofluorescence blocking solution (Coolaber, Beijing, China). Second antibody (1:2000) was added for incubation at 37℃ in dark for 1 hour. Then 4′,6-diamidino−2-phenylindole was added and the cells were incubated for 10 minutes. Images were captured with a Leica inverted fluorescent microscope and ImageJ software was used to analyze the fluorescence intensity. 2.9 Western blot After 24 hours of exposure to different doses of PES, the supernatant was collected, centrifuged, and the cells were lysed with protein lysis buffer after two rinses with ice-cold phosphate buffered saline (PBS). The Bradford protein assay kit, manufactured by Beyotime (Beijing, China), was utilized to accurately quantify protein concentrations. Protein was electrophoresed on 10% or 12.5% PAGE gels and then electrotransferred to PVDF membrane (Bio-Rad Laboratories, CA, USA). The membranes were incubated overnight at 4°C with specific primary antibodies for effective binding after blocking with fresh 5% nonfat milk in Tris-Buffered Saline (TBS) containing 0.5% Tween−20 and washing three times. After thorough washing, the membranes were incubated with suitable secondary antibodies for 1 hour at ambient temperature to enhance detection sensitivity. The expression of antibody-bound proteins was detected using ECL systems (Bio-Rad). 2.10 Statistical analysis Statistical analysis of the collected data was conducted utilizing GraphPad Prism 9.0 software from Hearne Scientific Software (Chicago, IL, USA). All presented data represent the mean ± SEM calculated from three independent experiments, ensuring reproducibility and accuracy. One-way analysis of variance (ANOVA) was performed for multiple comparisons. A statistically significant difference was defined as a value of P < 0.05. 3. Results 3.1 Comparison of heat shock protein 70 expression levels To investigate the expression level of HSP70 between tissues and serum ,which was measured by ELISA. As shown in Figure 1A, the expression level of HSP70 in the serum of HCC patients was significantly higher than normal people. Similarly, we performed immunohistochemical staining (Figure 1B) and immunofluorescence staining (Figure 1C) for HSP70 in cancerous and paracancerous tissues, respectively, and the results were consistent with the above experiments. It can be seen that HCC has a high level of HSP70 expression. 3.2 PES reduces cell viability and inhibits the proliferation of HCC cells As shown in Figure 2A, the differences in the expression levels of HSP70 in different cell lines, such as HepG2, Hep3B, and HUH-7, were examined, and it was found that the expression of HSP70 in HepG2 cells was higher compared with the other two groups, and it was eventually selected as the experimental cell. To assess the impact of PES on cell viability, HepG2 cells were subsequently exposed to either the vehicle control or varying concentrations of PES (ranging from 5 to 30 µM) for durations of 24 and 48 hours, respectively. CCK-8 assays found that PES induced loss of viability in HepG2 cells in a time - and dose-dependent manner, with 50% inhibitory concentrations (IC50) 12.5 and 8.8 µM for 24- and 48-hour PES treatment, respectively(Figure 2B). We wondered whether PES might be able to suppress the proliferation of HCC cells. As expected, treatment of HepG2 cells with different concentrations of PES, ranging from 5 to 25 µM, resulted in modest inhibition of EDU entry into the nucleus, indicating that PES interferes with the growth of HCC cells (Figure 2C).These results suggested that PES effectively reduced cell viability and inhibited the proliferation of HepG2 cells. 3.3 PES induces apoptosis in HCC cells Previous research has demonstrated a significantly elevated expression of HSP70 in HCC cells, in contrast to normal cell lines. Western blot was employed to detect the expression levels of HSP70 and apoptosis-related proteins to confirm the ability of PES to mediate apoptosis in HCC cells. HepG2 cells were treated with or without PES (10 to 40 µM) for 24 h. As shown in figure 3A , with increasing concentration of PES, we observed a gradual decrease in HSP70 expression. Furthermore, our study showed that PES increased the levels of the apoptotic proteins Bax, cleaved caspase-3, and cleaved caspase-9, while decreasing the expression of the anti-apoptotic protein Bcl-2. We found that PES increased the activity of Caspase-3 in the HepG2 cells(Figure 3B). Next, the expression of HSP70 and the apoptosis gene caspase-3 were compared in different treatment groups by immunofluorescence, as illustrated in figure 3C, in the control group, the cell was strongly fluorescent in red and blue, and the red fluorescent cell was significantly decreased with PES treatment, indicating that the HCC cells were treated with PES to reduce the expression of HSP70. In addition ,with increasing PES concentration, the expression of the pro-apoptotic protein Caspase-3 in HCC cells was found to gradually increase(Figure 3D). Overall, the drug was found to be an inhibitor of HSP70 expression and a promoter of apoptosis in HCC cells. 3.4 PES induces apoptosis in HCC cells by inhibiting the PI3K/AKT pathway The PI3K/AKT signaling pathway is widely recognized as one of the most common pathways involved in the development of cancer and has been the subject of extensive research. As shown in figure 4A, the levels of p-PI3K and p-AKT in HepG2 cells were decreased by treatment with PES. Next, to confirm whether PES induced apoptosis through the PI3K/AKT pathway, we treated the cells with a PI3K inhibitor (LY294002) prior to treating the cells with PES, and our experimental results showed that pretreatment with LY294002 enhanced the PES-induced reduction of p-AKT(Figure 4B). 4. Discussion Liver cancer is among the most pervasive malignant neoplasms worldwide, where HCC accounts for over 80% of primary liver cancers [15] . However, despite surgical resection, survival rates remain unsatisfactory due to the significantly high rates of metastasis and recurrence [16,17] . While non-alcoholic steatohepatitis linked to metabolic syndrome or diabetes mellitus is increasingly emerging as a prevalent risk factor in the West, infections with hepatitis B and hepatitis C viruses remain significant contributors to the development of hepatocellular carcinoma. All of these risk factors are potentially preventable, underscoring the immense potential of risk prevention in mitigating the global burden of hepatocellular carcinoma [11] . Therefore, there is an urgent need for the active search for effective pharmaceutical treatments and diagnostic strategies for the management of HCC. HSPs are involved in the stabilization of aberrantly expressed tumor-associated genes in tumorigenesis and play a role in enhancing anti-tumor immunity. Thus, there is growing evidence to suggest that HSPs may serve as promising targets for treating cancer [18,19] . The biological function of a small molecule HSP70 inhibitor, known as PES, has been shown to be effective against a variety of cancers [20,21,22] . PES has been shown to be capable of inducing programmed cell death in cancer cells, both in vivo and in vitro [20,21] . For instance, PES induces cell cycle arrest in the G2/M phase, apoptosis and inhibition of growth of oral squamous cell carcinoma cells [21] . PES has also demonstrated anti-tumor activity against human non-small cell lung cancer in vitro and in vivo [20] . In our current study, we have discovered that PES exhibits inhibitory effects on cell proliferation and induces apoptosis in HepG2 cells via modulation of the PI3K/AKT signaling pathway. Invasion and metastasis are critical characteristics of malignant tumors and are significantly associated with patient survival and prognosis. First of all, PES exhibited inhibitory effects on the viability of human HCC cells in a time- and dose-dependent manner. Hoechst 33342 staining data showed that after PES treatment of HepG2 cells for 24h, the ability of EDU to enter the cell nucleus was gradually inhibited with the increment of PES concentration compared with the control group, suggesting that PES could effectively inhibit the proliferation of HCC cells. PES can induce apoptosis in cells in addition to its growth inhibitory effect. Apoptosis is the process by which a cell stops growing and dividing and moves on to a process that ultimately leads to cell death [23] . The activation of cysteine-aspartic acid proteases holds a pivotal role in initiating the process of apoptosis. These proteases, referred to as caspases, have been identified as important factors in the programmed death of cells [24,25] . Therefore, for an accurate assessment of apoptosis, it is critical to evaluate changes in subsets of the caspase family. In this study, our results indicated that PES dose-dependently induced apoptosis. Western blot and quantitative RT-PCR results demonstrated a significant decrease in HSP70 expression in HepG2 cells following treatment with PES, which is consistent with its known biological function. In addition, exposure to PES treatment led to an upregulation of pro-apoptotic proteins, namely Bax and Caspase-3, alongside a downregulation of the anti-apoptotic protein Bcl-2. Immunofluorescent chemical staining further confirmed these results. From this it can be seen that PES may be a potential treatment strategy to induce apoptosis in HCC cells. Previous studies have shown that tumor development is closely linked to key molecules and signaling pathways, which are the basis of cancer [26] . The PI3K/AKT signaling pathway is widely recognized as one of the most prevalent pathways influencing cancer development and has been extensively studied as a potential target for cancer intervention and therapy [27,28] . The PI3K/AKT signaling pathway is the primary regulator of cell survival, growth, differentiation, metabolism, and reorganization of the cytoskeleton [29,30] . Most oncoproteins and tumor suppressors regulate cellular metabolism and signaling through the PI3K pathway, which plays a critical role in several human cancers, with dysregulation leading to activation and inactivation of related proteins [31,32] . Compounds specifically targeting the PI3K/AKT pathway have the potential to effectively induce apoptosis and thereby hinder the progression of tumors in cancer cells [33] . Our findings indicated that the levels of phosphorylated PI3K and AKT could be reduced by PES. Downregulation of phosphorylated AKT through a PI3K signaling pathway inhibitor led to an increase in HepG2 cell sensitivity to PES. These findings indicate that PES triggers apoptosis in HCC cells via the PI3K/AKT signaling pathway. In this study, we demonstrated that PES has the ability to suppress the activity and induce apoptosis in HCC cells in vitro. Additionally, the suppression of cellular viability induced by PES was linked to the modulation of HSP70 in human hepatocellular carcinoma cells. More importantly, our study demonstrated for the first time that PES, through inhibition of the PI3K/AKT signaling pathway, induced apoptosis in hepatocellular carcinoma cells. Taken together, all these findings provided new insights into the potential anti-tumor activity of PES and made it a promising agent for the treatment of human HCC. Declarations Acknowledgements Not applicable. Funding Taizhou People's Hospital Affiliated to Nanjing Medical University Research Fund (TZKY20230210）. Availability of data and materials The data generated in the present study may be requested from the corresponding author. CRediT authorship contribution statement Shengfeng Fu: Conceptualization, Data curation, Formal analysis, Writing - original draft. Haifeng Li: Methodology, Resources, Writing - review & editing. Xiaoqian Zhao: Methodology, Validation. Yu Zhang: Data Curation, Visualization. Jiali Chen: Data Curation, Resources. Changhe Zhang: Supervision, Funding acquisition. Yuxia Zhang: Supervision. Consent for publication All authors approved to publish the study in this journal. Competing interests The authors declare that they have no competing interests. Ethics approval and consent to participate Prior to the commencement of this study, all participants were informed and provided written informed consent. 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Supplementary Files SupplementaryInformation.pdf Cite Share Download PDF Status: Under Review Version 1 posted Reviewers invited by journal 27 Apr, 2026 Editor assigned by journal 09 Feb, 2026 Editor invited by journal 30 Jan, 2026 Submission checks completed at journal 23 Jan, 2026 First submitted to journal 23 Jan, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8501296","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":630312152,"identity":"aa7f7b7a-67b8-490c-a218-c31d9e8aa2d1","order_by":0,"name":"Shengfeng Fu","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Shengfeng","middleName":"","lastName":"Fu","suffix":""},{"id":630312154,"identity":"4c2f3b75-a513-4445-83c2-c638079a6f11","order_by":1,"name":"Haifeng Li","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Haifeng","middleName":"","lastName":"Li","suffix":""},{"id":630312156,"identity":"cfabd086-30db-4582-ab89-8b8b5d044f36","order_by":2,"name":"Xiaoqian Zhao","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Xiaoqian","middleName":"","lastName":"Zhao","suffix":""},{"id":630312157,"identity":"ce68f803-31bf-4fba-93de-ac9d9bf608cb","order_by":3,"name":"Yu Zhang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Yu","middleName":"","lastName":"Zhang","suffix":""},{"id":630312158,"identity":"b90d8f07-94b5-448c-b68f-58c2c95d9f2a","order_by":4,"name":"Jiali Chen","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Jiali","middleName":"","lastName":"Chen","suffix":""},{"id":630312159,"identity":"e9cc6b68-4a40-4aa6-8ba2-9dd6d879b6b9","order_by":5,"name":"Changhe Zhang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Changhe","middleName":"","lastName":"Zhang","suffix":""},{"id":630312160,"identity":"9016888d-807b-4a18-8848-0481ab8d348d","order_by":6,"name":"Yuxia Zhang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA2UlEQVRIiWNgGAWjYBACfmb+Dwc+VNjw8LM3HyBOi2R7g+HBGWfS5CR7jiUQp8XgzAHjw7xth40NbvgYEOmyGwkJh3nYDiduuMHz8cYbBjs53QYCOhhnJBw4OIcnPXHm7d7NlnMYko3NDhDQwiyR2HDgjYR1Yt+ds9ukeRgOJG4jpIVNIpnhAI8Bc2LDjZxnxGnh4TnGcJAnwdlY4EYOG3FaJNh7GA7OOAAOZGPLOQZE+MX+MA/zh4//wFH58MabCjs5glpQreQhNmqQtJCqYxSMglEwCkYEAACe8EpZkfqMZwAAAABJRU5ErkJggg==","orcid":"","institution":"","correspondingAuthor":true,"prefix":"","firstName":"Yuxia","middleName":"","lastName":"Zhang","suffix":""}],"badges":[],"createdAt":"2026-01-02 13:39:51","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8501296/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8501296/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":108615727,"identity":"4e9d4d60-850a-49e5-84f6-198503abf7a9","added_by":"auto","created_at":"2026-05-06 13:55:46","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":486318,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComparison of heat shock protein 70 expression levels. \u003c/strong\u003e(A) The expression of HSP70 protein in serum was determined by Elisa assay. (B, C) Levels of HSP70 protein in 6 groups of HCC tissues and paracancerous tissues were determined by IHC and IF staining. The values are presented as the means ±SEM at least three independent experiments, **** indicates p \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-8501296/v1/e0e9cce54609ae0b88c58d92.png"},{"id":108615542,"identity":"e61c9270-5c65-401f-b52a-8f538712164c","added_by":"auto","created_at":"2026-05-06 13:55:04","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":974779,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePES reduces cell viability and inhibits the proliferation of HCC cells.\u003c/strong\u003e (A) Comparison of HSP70 protein levels in three cell lines, HepG2, Hep3B and HUH-7 by western blot. (B) Cells were incubated with various concentrations (0, 5, 10, 15, 20, 25 and 30μM) of PES for 24h and 48h. Cell viability was determined by using CCK-8 assay. (C) Cells were incubated with various concentrations (0, 5, 10, 15, 20 and 25μM) of PES for 24h. The proliferation of cells was determined by Hoechst 33342 staining. The values are presented as the means ± SEM at least three independent experiments. \u003csup\u003e#\u003c/sup\u003e, \u003csup\u003e##\u003c/sup\u003e, and **** indicate p \u0026lt; 0.05, p \u0026lt; 0.01, and p \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-8501296/v1/354c5c42696a78391d907840.png"},{"id":108615445,"identity":"334245d6-616a-4dfb-8cc1-e7c377564cf0","added_by":"auto","created_at":"2026-05-06 13:54:38","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2151576,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePES induces apoptosis in HepG2 cells. Cells were incubated with indicated PES concentration for 24h.\u003c/strong\u003e (A) Apoptosis-related proteins and HSP70 protein expression levels were determined by western blot. (B) Caspase-3 activity was determined as described in material and methods. (C,D) Caspase-3 and HSP70 protein expression levels were determined by IF staining. The values are presented as the means ±SEM at least three independent experiments. *, **, ***, and **** indicate p \u0026lt; 0.05, p \u0026lt; 0.01, p \u0026lt; 0.001, and p \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Fig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-8501296/v1/0b38d634593f9a0994e56ed5.png"},{"id":108615749,"identity":"2633202b-cf8e-40fe-a56f-f308fffd54ff","added_by":"auto","created_at":"2026-05-06 13:55:50","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":293958,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePES induces apoptosis in HCC cells by inhibiting the PI3K/AKT pathway.\u003c/strong\u003e(A) Cells were incubated with various concentrations (0, 10, 20 and 40μM) of PES for 24h. The protein levels of p-PI3K, PI3K, p-Akt and Akt were determined by western blot. Cells were pretreated with LY294002 (10μM) for 2h and then incubated with PES (20μM) for 24h. (B) Cell lysates were subjected to western blot. The values are presented as the mean ±SEM of at least three independent experiments. *, **, and **** indicate p \u0026lt; 0.05, p \u0026lt; 0.01, and p \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Fig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-8501296/v1/7ef315d21fb9f7059e4135f8.png"},{"id":108615954,"identity":"9afb0fe5-3b83-46f8-b304-ac032aa2b353","added_by":"auto","created_at":"2026-05-06 13:56:38","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4027553,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8501296/v1/de8dcadb-9bee-4858-ae8f-aa8a577fef02.pdf"},{"id":108615444,"identity":"53b919d2-4f1a-49b4-80ca-301a98dfc102","added_by":"auto","created_at":"2026-05-06 13:54:38","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":679671,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformation.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8501296/v1/d22a5544ccb81e8d3c50bf5f.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"2-phenylacetylsulfonamide promotes apoptosis of hepatocellular carcinoma by inhibiting HSP70 through PI3K/AKT signaling pathway","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eHepatocellular carcinoma (HCC) is one of the major reasons for cancer-related mortality throughout the world, accounting for about 90% of all primary liver cancer cases\u003csup\u003e[1]\u003c/sup\u003e. Early diagnosis and treatment of HCC is virtually impossible due to the lack of specific clinical signs and symptoms\u003csup\u003e[2]\u003c/sup\u003e. Despite significant progress in treating HCC using modern surgical and chemotherapeutic techniques, the 5-year survival rate remains under 18%\u003csup\u003e[3]\u003c/sup\u003e. As such, new therapeutic agents and effective treatment strategies are urgently required to improve prognosis of HCC patients.\u003c/p\u003e\n\u003cp\u003eIn every living organism, heat shock proteins (HSPs) serve as an ancient defensive mechanism, acting as molecular chaperones to promote the appropriate folding and refolding of misfolded proteins, as well as assisting in the breakdown of damaged and aging cells\u003csup\u003e[4,5]\u003c/sup\u003e. HSP70 is a protein associated with tumorigenicity and possessing various functions, such as performing as a cellular safeguard, and exhibiting anti-apoptotic and immune functions\u003csup\u003e[6]\u003c/sup\u003e. Increasingly, the expression of HSP70 has been shown to be significantly elevated in human tumor cells and may influence cancer development through a variety of mechanisms. In contrast, inhibiting or knocking down HSP70 can reduce tumor size and even lead to complete tumor regression\u003csup\u003e[7,8]\u003c/sup\u003e. An attractive strategy for cancer therapy is therefore to target HSP70.\u003c/p\u003e\n\u003cp\u003e2-phenylacetylsulfonamide (also referred to as pifithrin-\u0026micro; or PES), a selective inhibitor of HSP70, was initially thought to be an inhibitor of p53 binding to mitochondria\u003csup\u003e[9]\u003c/sup\u003e. Mechanically, PES reacts covalently with the sulfhydryl group on HSP70, leading to disruption of the binding between HSP70\u0026apos;s co-chaperone and the substrate protein, resulting in inhibition of its activity\u003csup\u003e[10,11]\u003c/sup\u003e. PES-induced cancer cell death is primarily associated with the accumulation of misfolded proteins, disruption of autophagy and impairment of lysosomal function\u003csup\u003e[12,13,14]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;In this study, we aimed to look into the connection between PES, HSP70, and apoptosis-related genes in HCC cells. The findings exhibited that PES suppressed the expression of HSP70 and triggered apoptosis. In this process, we focused on determining the expression of apoptotic genes such as Bax, Bcl-2, Caspase-3, and Caspase-9. In addition, we confirmed that PES induces apoptosis through the inhibition of the PI3K/AKT pathway.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003e2.1 Reagents and antibodies\u003c/h2\u003e\n \u003cp\u003ePES and LY294002 were purchased from Med Chem Express (MCE, Shanghai, China). Antibodies for HSP70 (1:1000), Bax (1:1000), Bcl-2 (1:1000), Caspase-3 (1:500) and GAPDH (1:10000) were obtained from Proteintech (Wuhan, China). Antibody for PI3K (1:1000), P-PI3K (1:1000), AKT (1:1000), P-AKT (1:1000), cleaved Caspase-3 (1:1000) and cleaved Caspase-9 (1:1000) were obtained from Cell Signal Technology (Boston, USA). The secondary antibodies were horseradish-peroxidase-conjugated secondary anti-mouse IgG or anti-rabbit IgG (Proteintech, Wuhan, China).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n \u003ch2\u003e2.2 Cell culture and treatment\u003c/h2\u003e\n \u003cp\u003eThe HCC cell lines, HepG2, Hep3B, HUH-7 were purchased from Procell (Wuhan, China). Cell culture were performed in DMEM supplemented with 10% Every Green fetal bovine serum (Zhejiang, China) and 1% (v/v) penicillin/ streptomycin and placed in a humidified atmosphere with 5% CO2 at 37 ℃. PES was solubilized with DMSO (Sigma, St. Louis, MO, USA).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n \u003ch2\u003e2.3 Determination of HSP70\u003c/h2\u003e\n \u003cp\u003eBlood samples were collected from 6 patients with clinically diagnosed hepatocellular carcinoma (experimental group) and 6 normal people (control group). After centrifugation, the upper layer of serum was taken and stored in a refrigerator at -80℃. Six cases of liver cancer tissues and adjacent tissues were collected and stored in a refrigerator at -80℃ under liquid nitrogen. Then the expression of HSP70 in serum and tissue was detected by ELISA kit, immunohistochemistry and Immunofluorescence. The design and reporting of this study comply with the requirements of the Declaration of Helsinki and have been approved by the Clinical Research Ethics Committee of Taizhou People\u0026apos;s Hospital. We confirm that all research was conducted in accordance with relevant guidelines and regulations, and informed consent was obtained from all participants and/or their legal guardians.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n \u003ch2\u003e2.4 Screening of HCC cell lines\u003c/h2\u003e\n \u003cp\u003eThree cell lines, HepG2, Hep3B and HUH-7, were selected as test subjects based on their different expression levels of HSP70, and then the content of HSP70 in these three cells was determined.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n \u003ch2\u003e2.5 Cell viability assay\u003c/h2\u003e\n \u003cp\u003eTo determine cell viability, the Cell Counting Kit-8 (CCK-8) assay kit was utilized. In brief, HepG2 cells were incubated overnight in 96-well plates, maintained at a cell density of 6\u0026times;10\u003csup\u003e3\u003c/sup\u003e per 100 \u0026micro;l. After 24 and 48 hours of treatment with different concentrations of PES, 10\u0026micro;l CCK-8 solution was added to each well, followed by incubation at 37 ℃ for a duration of 2 hours. The absorbance was accurately measured using a microplate reader (Biotek) specifically set at a wavelength of 450 nm.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003e2.6 Edu incorporation assay\u003c/h2\u003e\n \u003cp\u003eThe capacity of cell proliferation was evaluated through the 5-ethynyl\u0026minus;2-deoxyuridine (EdU) incorporation assay, which was conducted after subjecting the cells to 24 hours of treatment with diverse concentrations of PES, following the manufacturer\u0026apos;s instructions (Beyotime, Shanghai, China). The images were observed and recorded using an inverted fluorescence microscope (Leica).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\n \u003ch2\u003e2.7 Caspase\u0026minus;3 activity assay\u003c/h2\u003e\n \u003cp\u003eThe Caspase\u0026minus;3 Activity Kit (Beyotime Institute of Biotechnology, Guangzhou, China) was utilized to measure Caspase\u0026minus;3 activity, strictly adhering to the manufacturer\u0026apos;s protocol. This method is based on the ability of caspase\u0026minus;3 to catalyze the production of yellow pNA from the substrate acetyl-Asp-GluVal-Asp-p-nitroanilide (Ac-DEVD-pNA), and therefore the activity of caspase\u0026minus;3 can be detected by measuring the absorbance. After treatment, cells were harvested and lysed with lysis buffer. Lysates, assay buffer, and caspase\u0026minus;3 substrate were incubated together at a temperature of 37\u0026deg;C for a duration of 2 hours. Subsequently, the absorbance of the samples was accurately measured at a wavelength of 405 nm.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n \u003ch2\u003e2.8 Immunofluorescence\u003c/h2\u003e\n \u003cp\u003eCells were seeded at a density of 8 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells per well within 12-well plates. After 24 hours of treatment with various concentrations of PES, the cells were immobilized with a solution containing 4% paraformaldehyde. The cells were incubated with HSP70 (1:200) and Caspase\u0026minus;3 (1:500) overnight at 4℃ after blocking with immunofluorescence blocking solution (Coolaber, Beijing, China). Second antibody (1:2000) was added for incubation at 37℃ in dark for 1 hour. Then 4\u0026prime;,6-diamidino\u0026minus;2-phenylindole was added and the cells were incubated for 10 minutes. Images were captured with a Leica inverted fluorescent microscope and ImageJ software was used to analyze the fluorescence intensity.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003e2.9 Western blot\u003c/h2\u003e\n \u003cp\u003eAfter 24 hours of exposure to different doses of PES, the supernatant was collected, centrifuged, and the cells were lysed with protein lysis buffer after two rinses with ice-cold phosphate buffered saline (PBS). The Bradford protein assay kit, manufactured by Beyotime (Beijing, China), was utilized to accurately quantify protein concentrations. Protein was electrophoresed on 10% or 12.5% PAGE gels and then electrotransferred to PVDF membrane (Bio-Rad Laboratories, CA, USA). The membranes were incubated overnight at 4\u0026deg;C with specific primary antibodies for effective binding after blocking with fresh 5% nonfat milk in Tris-Buffered Saline (TBS) containing 0.5% Tween\u0026minus;20 and washing three times. After thorough washing, the membranes were incubated with suitable secondary antibodies for 1 hour at ambient temperature to enhance detection sensitivity. The expression of antibody-bound proteins was detected using ECL systems (Bio-Rad).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003e2.10 Statistical analysis\u003c/h2\u003e\n \u003cp\u003eStatistical analysis of the collected data was conducted utilizing GraphPad Prism 9.0 software from Hearne Scientific Software (Chicago, IL, USA). All presented data represent the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM calculated from three independent experiments, ensuring reproducibility and accuracy. One-way analysis of variance (ANOVA) was performed for multiple comparisons. A statistically significant difference was defined as a value of P\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"3. Results","content":"\u003cp\u003e3.1 Comparison of heat shock protein 70 expression levels\u003c/p\u003e\n\u003cp\u003eTo investigate the expression level of HSP70 between tissues and serum ,which was measured by ELISA. As shown in Figure 1A, the expression level of HSP70 in the serum of HCC patients was significantly higher than normal people. Similarly, we performed immunohistochemical staining (Figure 1B) and immunofluorescence staining (Figure 1C) for HSP70 in cancerous and paracancerous tissues, respectively, and the results were consistent with the above experiments. It can be seen that HCC has a high level of HSP70 expression.\u003c/p\u003e\n\u003cp\u003e3.2 PES reduces cell viability and inhibits the proliferation of HCC cells\u003c/p\u003e\n\u003cp\u003eAs shown in Figure 2A, the differences in the expression levels of HSP70 in different cell lines, such as HepG2, Hep3B, and HUH-7, were examined, and it was found that the expression of HSP70 in HepG2 cells was higher compared with the other two groups, and it was eventually selected as the experimental cell.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo assess the impact of PES on cell viability, HepG2 cells were subsequently exposed to either the vehicle control or varying concentrations of PES (ranging from 5 to 30 \u0026micro;M) for durations of 24 and 48 hours, respectively. CCK-8 assays found that PES induced loss of viability in HepG2 cells in a time - and dose-dependent manner, with 50% inhibitory concentrations (IC50) 12.5 and 8.8 \u0026micro;M for 24- and 48-hour PES treatment, respectively(Figure 2B).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe wondered whether PES might be able to suppress the proliferation of HCC cells. As expected, treatment of HepG2 cells with different concentrations of PES, ranging from 5 to 25 \u0026micro;M, resulted in modest inhibition of EDU entry into the nucleus, indicating that PES interferes with the growth of HCC cells (Figure 2C).These results suggested that PES effectively reduced cell viability and inhibited the proliferation of HepG2 cells.\u003c/p\u003e\n\u003cp\u003e3.3 PES induces apoptosis in HCC cells\u003c/p\u003e\n\u003cp\u003ePrevious research has demonstrated a significantly elevated expression of HSP70 in HCC cells, in contrast to normal cell lines. Western blot was employed to detect the expression levels of HSP70 and apoptosis-related proteins to confirm the ability of PES to mediate apoptosis in HCC cells. HepG2 cells were treated with or without PES (10 to 40 \u0026micro;M) for 24 h. As shown in figure 3A , with increasing concentration of PES, we observed a gradual decrease in HSP70 expression. Furthermore, our study showed that PES increased the levels of the apoptotic proteins Bax, cleaved caspase-3, and cleaved caspase-9, while decreasing the expression of the anti-apoptotic protein Bcl-2.\u003c/p\u003e\n\u003cp\u003eWe found that PES increased the activity of Caspase-3 in the HepG2 cells(Figure 3B). Next, the expression of HSP70 and the apoptosis gene caspase-3 were compared in different treatment groups by immunofluorescence, as illustrated in figure 3C, in the control group, the cell was strongly fluorescent in red and blue, and the red fluorescent cell was significantly decreased with PES treatment, indicating that the HCC cells were treated with PES to reduce the expression of HSP70. In addition ,with increasing PES concentration, the expression of the pro-apoptotic protein Caspase-3 in HCC cells was found to gradually increase(Figure 3D). Overall, the drug was found to be an inhibitor of HSP70 expression and a promoter of apoptosis in HCC cells.\u003c/p\u003e\n\u003cp\u003e3.4 PES induces apoptosis in HCC cells by inhibiting the PI3K/AKT pathway\u003c/p\u003e\n\u003cp\u003eThe PI3K/AKT signaling pathway is widely recognized as one of the most common pathways involved in the development of cancer and has been the subject of extensive research. As shown in figure 4A, the levels of p-PI3K and p-AKT in HepG2 cells were decreased by treatment with PES. Next, to confirm whether PES induced apoptosis through the PI3K/AKT pathway, we treated the cells with a PI3K inhibitor (LY294002) prior to treating the cells with PES, and our experimental results showed that pretreatment with LY294002 enhanced the PES-induced reduction of p-AKT(Figure 4B).\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eLiver cancer is among the most pervasive malignant neoplasms worldwide, where HCC accounts for over 80% of primary liver cancers\u003csup\u003e[15]\u003c/sup\u003e. However, despite surgical resection, survival rates remain unsatisfactory due to the significantly high rates of metastasis and recurrence\u003csup\u003e[16,17]\u003c/sup\u003e. While non-alcoholic steatohepatitis linked to metabolic syndrome or diabetes mellitus is increasingly emerging as a prevalent risk factor in the West, infections with hepatitis B and hepatitis C viruses remain significant contributors to the development of hepatocellular carcinoma. All of these risk factors are potentially preventable, underscoring the immense potential of risk prevention in mitigating the global burden of hepatocellular carcinoma\u003csup\u003e[11]\u003c/sup\u003e. Therefore, there is an urgent need for the active search for effective pharmaceutical treatments and diagnostic strategies for the management of HCC. HSPs are involved in the stabilization of aberrantly expressed tumor-associated genes in tumorigenesis and play a role in enhancing anti-tumor immunity. Thus, there is growing evidence to suggest that HSPs may serve as promising targets for treating cancer\u003csup\u003e[18,19]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThe biological function of a small molecule HSP70 inhibitor, known as PES, has been shown to be effective against a variety of cancers\u003csup\u003e[20,21,22]\u003c/sup\u003e. PES has been shown to be capable of inducing programmed cell death in cancer cells, both in vivo and in vitro\u003csup\u003e[20,21]\u003c/sup\u003e. For instance, PES induces cell cycle arrest in the G2/M phase, apoptosis and inhibition of growth of oral squamous cell carcinoma cells\u003csup\u003e[21]\u003c/sup\u003e. PES has also demonstrated anti-tumor activity against human non-small cell lung cancer in vitro and in vivo\u003csup\u003e[20]\u003c/sup\u003e. In our current study, we have discovered that PES exhibits inhibitory effects on cell proliferation and induces apoptosis in HepG2 cells via modulation of the PI3K/AKT signaling pathway.\u003c/p\u003e\n\u003cp\u003eInvasion and metastasis are critical characteristics of malignant tumors and are significantly associated with patient survival and prognosis. First of all, PES exhibited inhibitory effects on the viability of human HCC cells in a time- and dose-dependent manner. Hoechst 33342 staining data showed that after PES treatment of HepG2 cells for 24h, the ability of EDU to enter the cell nucleus was gradually inhibited with the increment of PES concentration compared with the control group, suggesting that PES could effectively inhibit the proliferation of HCC cells. PES can induce apoptosis in cells in addition to its growth inhibitory effect. Apoptosis is the process by which a cell stops growing and dividing and moves on to a process that ultimately leads to cell death\u003csup\u003e[23]\u003c/sup\u003e. The activation of cysteine-aspartic acid proteases holds a pivotal role in initiating the process of apoptosis. These proteases, referred to as caspases, have been identified as important factors in the programmed death of cells\u003csup\u003e[24,25]\u003c/sup\u003e. Therefore, for an accurate assessment of apoptosis, it is critical to evaluate changes in subsets of the caspase family. In this study, our results indicated that PES dose-dependently induced apoptosis. Western blot and quantitative RT-PCR results demonstrated a significant decrease in HSP70 expression in HepG2 cells following treatment with PES, which is consistent with its known biological function. In addition, exposure to PES treatment led to an upregulation of pro-apoptotic proteins, namely Bax and Caspase-3, alongside a downregulation of the anti-apoptotic protein Bcl-2. Immunofluorescent chemical staining further confirmed these results. From this it can be seen that PES may be a potential treatment strategy to induce apoptosis in HCC cells.\u003c/p\u003e\n\u003cp\u003ePrevious studies have shown that tumor development is closely linked to key molecules and signaling pathways, which are the basis of cancer\u003csup\u003e[26]\u003c/sup\u003e. The PI3K/AKT signaling pathway is widely recognized as one of the most prevalent pathways influencing cancer development and has been extensively studied as a potential target for cancer intervention and therapy\u003csup\u003e[27,28]\u003c/sup\u003e. The PI3K/AKT signaling pathway is the primary regulator of cell survival, growth, differentiation, metabolism, and reorganization of the cytoskeleton\u003csup\u003e[29,30]\u003c/sup\u003e. Most oncoproteins and tumor suppressors regulate cellular metabolism and signaling through the PI3K pathway, which plays a critical role in several human cancers, with dysregulation leading to activation and inactivation of related proteins\u003csup\u003e[31,32]\u003c/sup\u003e. Compounds specifically targeting the PI3K/AKT pathway have the potential to effectively induce apoptosis and thereby hinder the progression of tumors in cancer cells\u003csup\u003e[33]\u003c/sup\u003e. Our findings indicated that the levels of phosphorylated PI3K and AKT could be reduced by PES. Downregulation of phosphorylated AKT through a PI3K signaling pathway inhibitor led to an increase in HepG2 cell sensitivity to PES. These findings indicate that PES triggers apoptosis in HCC cells via the PI3K/AKT signaling pathway.\u003c/p\u003e\n\u003cp\u003eIn this study, we demonstrated that PES has the ability to suppress the activity and induce apoptosis in HCC cells in vitro. Additionally, the suppression of cellular viability induced by PES was linked to the modulation of HSP70 in human hepatocellular carcinoma cells. More importantly, our study demonstrated for the first time that PES, through inhibition of the PI3K/AKT signaling pathway, induced apoptosis in hepatocellular carcinoma cells. Taken together, all these findings provided new insights into the potential anti-tumor activity of PES and made it a promising agent for the treatment of human HCC.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTaizhou People\u0026apos;s Hospital Affiliated to Nanjing Medical University Research Fund (TZKY20230210）.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data generated in the present study may be requested from the corresponding author.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eCRediT authorship contribution statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eShengfeng Fu: Conceptualization, Data curation, Formal analysis, Writing - original draft. Haifeng Li: Methodology, Resources, Writing - review \u0026amp; editing. Xiaoqian Zhao: Methodology, Validation. Yu Zhang: Data Curation, Visualization. Jiali Chen: Data Curation, Resources. Changhe Zhang: Supervision, Funding acquisition. Yuxia Zhang: Supervision.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors approved to publish the study in this journal.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePrior to the commencement of this study, all participants were informed and provided written informed consent. Fresh serums and HCC tissues were obtained from the Taizhou people\u0026apos;s Hospital, Nanjing Medical University and approved by the Clinical Research Ethics Committee of Taizhou People\u0026apos;s Hospital (Taizhou, China; NO: KY-2023-013-01).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eG. Maciej,W. Krzysztof,K. Leszeket al., Milestones in the treatment of hepatocellular carcinoma: A systematic review., Crit. Rev. Oncol. Hematol. ｜157｜(0)｜(2020)10.1016/j.critrevonc.2020.103179\u003c/li\u003e\n\u003cli\u003eF. Meili,W. Fuqiang,L. Zhenglinget al., 4SC-202 activates ASK1-dependent mitochondrial apoptosis pathway to inhibit hepatocellular carcinoma cells., Biochem. Biophys. Res. Commun. ｜471｜(2)｜(2016)10.1016/j.bbrc.2016.01.030\u003c/li\u003e\n\u003cli\u003eS. Rebecca L,M. Kimberly D,J. Ahmedin, Cancer statistics, 2019., CA. Cancer. J. Clin. ｜69｜(1)｜(2019)10.3322/caac.21551\u003c/li\u003e\n\u003cli\u003eK. Harm H,C. 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Xuet al., Effect of evodiagenine mediates photocytotoxicity on human breast cancer cells MDA-MB-231 through inhibition of PI3K/AKT/mTOR and activation of p38 pathways., Fitoterapia. ｜99｜(0)｜(2014)10.1016/j.fitote.2014.10.010\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Hepatocellular carcinoma, HSP70, PES, Apoptosis","lastPublishedDoi":"10.21203/rs.3.rs-8501296/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8501296/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eHeat shock proteins (HSPs) are found widely across various organisms and play a crucial role in tumor and malignancy formation. Among all HSPs, HSP70 is the most extensively researched. 2-phenylacetylsulfonamide (PES) is a covalent inhibitor of HSP70, which has a significant effect on inhibiting wide range of cancer cells. Nevertheless, the effects of PES on hepatocellular carcinoma (HCC) cells are still largely unknown. The objective of this research was to investigate the relationship between PES and apoptosis-related proteins in human HCC, and to assess the potential mechanism of drug action on HCC. This study demonstrated the anti-cancer activity of PES against HCC cells. Cell viability was assessed utilizing the CCK-8 kit, which revealed that PES suppressed the growth of HepG2 cells in a dose-and time-dependent. Furthermore, the data from Hoechst 33342 staining indicate a gradual suppression of HCC cell proliferation with increasing PES concentration. The expression of the target gene HSP70 and various apoptotic genes was quantified using Western blot analysis. The findings demonstrate that PES significantly inhibited the expression of HSP70 and promoted the apoptosis of human HepG2 cells. Moreover, the immunofluorescence findings indicated a notable reduction in the expression of the target gene HSP70 in PES-treated cells of HCC, whereas the expression of Caspase-3 was up-regulated. Furthermore, our findings demonstrated that PES induces apoptosis in HepG2 cells by suppressing the PI3K/AKT signaling pathway. These results demonstrated that PES has anti-tumor activity against human HCC and may be an effective drug for HCC treatment.\u003c/p\u003e","manuscriptTitle":"2-phenylacetylsulfonamide promotes apoptosis of hepatocellular carcinoma by inhibiting HSP70 through PI3K/AKT signaling pathway","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-06 13:53:19","doi":"10.21203/rs.3.rs-8501296/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewersInvited","content":"","date":"2026-04-27T12:02:38+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-09T09:05:09+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-01-30T13:09:44+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-01-24T01:54:45+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2026-01-24T01:50:08+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"30ff1839-e687-4290-91e2-dd131e794b22","owner":[],"postedDate":"May 6th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":67086963,"name":"Biological sciences/Cancer"},{"id":67086964,"name":"Biological sciences/Cell biology"},{"id":67086965,"name":"Biological sciences/Drug discovery"},{"id":67086966,"name":"Biological sciences/Molecular biology"},{"id":67086967,"name":"Health sciences/Oncology"}],"tags":[],"updatedAt":"2026-05-06T13:53:19+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-06 13:53:19","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8501296","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8501296","identity":"rs-8501296","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}