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. 2025 Jan 28;41(1):35. doi: 10.1007/s10565-024-09979-x

Targeting p38γ synergistically enhances sorafenib-induced cytotoxicity in hepatocellular carcinoma

Chen Huang 1,2, Chenliang Zhang 1,2, Jiajin Li 3, Yichun Duan 2, Qiulin Tang 1, Feng Bi 1,2,✉
PMCID: PMC11772449  PMID: 39871031

Abstract

Sorafenib (Sora) is a first-line treatment for patients with advanced hepatocellular carcinoma (HCC). It can significantly improve the survival rate of patients with advanced HCC, but it is prone to drug resistance during treatment, so the therapeutic effect is extremely limited. Here, we demonstrate that an elevated expression of protein kinase p38γ in hepatocellular carcinoma cells diminishes the tumor cells’ sensitivity to Sora. Pirfenidone (PFD) can augment Sora’s inhibitory effect on hepatocellular carcinoma by specifically targeting p38γ. Our study further uncovers that pirfenidone can synergistically boost the anti-hepatocellular carcinoma impact of Sora by impeding the autophagy heightened by p38γ. Taken together, our findings suggest that pirfenidone can work in concert with Sora to intensify its anti-tumor effect on hepatocellular carcinoma, thereby offering a novel therapeutic approach for Sora-mediated tumor treatment.

Graphical Abstract

PFD is an inhibitor of p38γ, which may affect the Beclin pathway by inhibiting the function of p38γ, thereby inhibiting the level of autophagy, and ultimately achieving the effect of sensitizing Sora. Created with BioRender.com

Graphical Headlights

  1. Knockdown of p38γ sensitized the inhibitory effect of Sora on HCC cells;

  2. PFD synergistically enhances the anti-HCC effect of Sora by targeting p38γ;

  3. PFD sensitizes the anti-HCC effect of Sora by inhibiting autophagy.

graphic file with name 10565_2024_9979_Figa_HTML.jpg

Supplementary Information

The online version contains supplementary material available at 10.1007/s10565-024-09979-x.

Keywords: Hepatocellular carcinoma, Sorafenib, P38γ, Autophagy, Cell drug resistance

Introduction

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors and the second most lethal cancer in the world, accounting for 90% of primary liver cancer (Llovet et al., 2016; Jemal et al., 2011; Ferlay et al., 2010). About 700,000 people died of liver cancer in 2012 (Torre et al., 2015). The World Health Organization estimates that more than 1 million people will die of liver cancer in 2030 (Geneva 2016). Due to the low detection rate of liver cancer in the curable stage and the high recurrence rate, the prognosis is poor (Liu et al., 2017). Despite recent advances in treatment strategies, the survival rate of patients is still unsatisfactory. Tumor resection or liver transplantation can treat liver cancer (especially early liver cancer). However, due to the high recurrence rate of liver cancer, only 20% of patients with early liver cancer can choose surgical treatment, and there is no sufficient and effective treatment for patients with advanced, metastatic or drug-resistant liver cancer (Ma et al., 2015; Sheng et al., 2018; Chen et al., 2014). Therefore, it is urgent to elucidate the mechanism of occurrence, metastasis and drug resistance of liver cancer and to carry out innovative systematic treatment for patients with advanced diseases. The occurrence of liver cancer involves a variety of cellular mechanisms, such as cell cycle and apoptosis dysfunction, inflammation-related molecular pathways and fibrosis processes. These mechanisms in turn represent important molecular targets for the development of new drug therapies (Jindal et al., 2019).

Sorafenib can be used to treat liver cancer (especially advanced liver cancer) and is one of the two first-line drugs approved by the Food and Drug Administration of the United States (Villanueva and Llovet 2011; Llovet et al., 2008). As a systematic regimen, Sorafenib significantly prolongs the survival time and improves the survival rate of patients with advanced liver cancer. Sora is a multi-kinase inhibitor (Llovet and Hernandez-Gea 2014) and RAF-MEK-ERK / MAPK pathway inhibitor. Existing studies have shown that Sorafenib can affect cell proliferation and survival, angiogenesis and other processes in vivo, thereby inhibiting tumor cell proliferation and inducing tumor cell apoptosis. (Raza and Sood 2014). Sora has been shown to exert anti-tumor effects through a variety of cell signal transduction pathways (Yang et al., 2021; Liu et al., 2019; Mazzoccoli et al., 2016). Although Sora has been shown to significantly improve overall survival, it cannot reverse disease progression due to resistance to anti-proliferative therapy. For some patients who seem to respond to Sora, it is still ineffective due to slow body response leading to tumor metastasis and progression (Villanueva and Llovet 2011). Therefore, overcoming drug resistance and enhancing tumor killing ability are the key to improving the therapeutic effect of Sora and expanding the population of benefiting patients. Combination therapy is an effective strategy to improve the effect of tumor targeted therapy.

p38 MAPKs is a cascade kinase involved in the control of cell response to cytokines and stress. There are four subtypes in mammals: p38α (MAPK14), β (MAPK11), γ (MAPK12) and δ (MAPK13). Different p38 subtypes may have opposite functions (Matesanz et al., 2018). In 2019, Sabio G (Kozono et al., 2013) et al. confirmed that p38MAPKγ (p38γ) acts as a CDK-like kinase, thereby cooperating with CDKs to regulate entry into the cell cycle. p38γ is an important driver of hepatocellular carcinoma, which promotes the proliferation of tumor cells by phosphorylating the cell cycle regulatory protein Rb. The potential of p38γ as a therapeutic target for liver cancer and may provide a new way for the treatment of liver cancer.

Pirfenidone (PFD) is a pyridine oral small molecule drug with anti-fibrosis, anti-oxidation and anti-inflammatory effects. It has been approved by PFD for clinical and preclinical studies to treat lung and liver fibrosis (Noble et al., 2011; Taniguchi et al., 2010; Carter, 2011; Lopez-De, et al., 2015; Miura et al., 2018; GARCíA L, HERNáNDEZ I, SANDOVAL A,, et al., 2002). PFD has a wide range of anti-fibrosis effects. Current multi-party studies have confirmed that PFD can have a good and extensive anti-fibrosis effect, and the reason may be that it acts on a variety of cytokines and inhibits their functions. These cytokines also include some transforming growth factors (such as TGF-β), tumor necrosis factor (such as TNF-α) and so on. and inhibit fibroblast proliferation and collagen synthesis, scavenging reactive oxygen species (Giri et al., 1999; Oku et al., 2008). Recently, more and more studies have shown that PFD has a certain anti-tumor effect. Studies have observed that PFD treatment reduces the incidence of lung cancer in patients with idiopathic pulmonary fibrosis (IPF) (Miura et al., 2018), and a number of in vitro studies support this observation. After exposure to PFD, the proliferation, differentiation and migration of non-small cell lung cancer (NSCLC) cells, mesothelioma and pancreatic cancer cells (Kozono et al., 2013; Li et al., 2018; Fujiwara et al., 2017) decreased. In vivo studies have also confirmed these findings that PFD alone or in combination with cisplatin significantly reduced the growth of lung cancer and pancreatic cancer cells by inducing changes in the tumor microenvironment (Kozono et al., 2013; Mediavilla-Varela et al., 2016). Although the existing research results can preliminarily confirm that pirfenidone can inhibit the proliferation of some tumor cells (Zou et al., 2017; Usugi et al., 2019; Lee et al., 1998), reduce the levels of hyaluronic acid and collagen in ECM and increase the pathway of chemotherapeutic drugs into tumors (Polydorou et al., 2017), and normalize the tumor microenvironment, the mechanism of PFD anti-tumor effect is still unclear. Recent studies have revealed that PFD is a specific inhibitor of p38γ(Aimo et al., 2022).

This study explored the function of p38γ in the treatment of hepatocellular carcinoma with Sora, and showed that p38γ reduced the sensitivity of hepatocellular carcinoma cells to Sora, and pirfenidone could synergistically sensitize Sora to hepatocellular carcinoma by targeting p38γ. Further studies have found that targeting p38γ sensitizes Sora by reducing the autophagy level of HCC. We combined pirfenidone with Sora to explore its inhibitory effect on hepatocellular carcinoma, and explored the synergistic anticancer mechanism of pirfenidone, in order to solve the drug resistance problem in the practical clinical application of Sora, and to further optimize the treatment plan for patients with advanced liver cancer.

Materials and methods

Materials

Sorafenib (CSNpharm, CSN10381), Pirfenidone (MCE, HY-B0673), Lipofectamine 2000 (Invitrogen, 11,668,500), DAPI (Invitrogen, 62,248), Ferrostatin-1 (MCE, HY-100579), SP600125 (MCE, HY-12041), Birinapant (MCE, HY-16591), Belnacasan (MCE, HY-13205), Wortmannin (MCE, HY-10197), SBI-0206965 (MCE, HY-16966), Bafilomycin A1 (MCE, HY-100558), Protein A/G Magnetic Beads (MCE, HY-K0202). Antibodie: p38 gamma/MAPK12 (Abcam, ab205926), GAPDH (Zen Bioscience, 200,306), LC3B (Abcam, ab192890), p62 (Abcam, ab109012), Phospho-p38 MAPK (Thr180/Tyr182) (CST, 9211), FLAG tag (Prospec, ANT-146), ATG5 (Proteintech, 10181–2-AP).

Cell culture

HepG2, Huh-7, PLC/PRF/5, BEL-7404, AML-12 and HEK293 cells (ATCC, Manassas, Virginia, USA), fetal bovine serum (FBS; Gibco / Invitrogen, USA), Dulbecco modified Eagle medium (DMEM; Gibco, USA). All cells were cultured in a medium containing 10% carbon dioxide at 37° C in a 5% carbon dioxide environment. STR was identified in all cells without mycoplasma contamination.

Cell viability assays

All cells were cultured in 96-well plates and detected by cell counting kit after culture or experimental treatment. In this study, cell counting kit-8 (Dojingdo, Kumamoto, Japan) was used to measure the relative growth of cells after transfection or treatment with specific drugs. The CCK-8 working solution is usually added to the DMEM medium (1:10), and 50 μl of the mixture is added to each well to be tested. The 96-well plates were placed in an incubator at 37° C for 1 h until the color became orange. The absorbance at 450 nm was read by a microplate reader. Usually, the OD value of the control group was 1 as the end time, and the data were collected and statistically analyzed.

Colony formation assays

The cells to be treated were inoculated in a 6-well plate and cultured in DMEM medium supplemented with FBS. After the cell growth was stable, the experimental treatment was performed. During the experiment, the cell state was observed every 2 days (at least) and the medium was replaced at the appropriate time, and the cells were placed in a cell incubator and continued to be cultured until the end of the experiment. Usually 7–14 days. After the experiment, fixed with paraformaldehyde, and then stained with crystal violet, and photographed and analyzed with the corresponding equipment.

SynergyFinder online analysis of drug interactions

After completing the cell CCK-8 experiment, the results of the CCK-8 experiment were filled in according to the Excel template provided by the website (https://synergyfinder.fimm.fi/). The information must contain the following information: PairIndex, Response, Drug 1, Drug 2, Concentration 1, Concentration 2 and Concentration unit. After uploading the data file, specify a specific type (survival rate or inhibition rate), then analyze and calculate, and save the analysis report and results. The SynergyFinder website provides four different reference models (HSA, Bliss, Loewe and ZIP), which are briefly summarized as follows: HAS is the simplest model, that is, the maximum value of a single drug effect at the corresponding concentration. The Bliss independent model assumes that the onset process of different drugs is independent and random, which is suitable for two drugs with different targets. The Loewe additive model assumes that the two drugs are the same to calculate the expected effect. The Zero interaction potency (ZIP) model combines the advantages of the Bliss model and the Loewe model, and is more suitable for systematic evaluation of high-throughput drug combination screening. Results Interpretation: The synergy score is generally understood as the average degree of excess due to drug interactions. For example, if the synergy score is 10, it corresponds to 10% of the expected effect. Although there is no specific value as a standard at present, the interpretation standard of synergy score can be determined according to experience: less than −10, there may be antagonism between the two drugs; from − 10 to 10, there may be a simple addition/subtraction effect between the two drugs; more than 10, there may be a synergistic effect between the two drugs. If the score is closer to 0, the evidence supporting the correlation between the two is more limited, and it can be considered that there is no drug interaction between the two, that is, it is irrelevant.

Transfection

The siRNA and plasmid transfection were carried out with Lipofectamine 2000 (Invitrogen) according to the manufacturer 's instructions. The siRNAs were synthesized by Ribobio. The siRNA sequences used in this study are as follows: siMapk12_001: 5′-CAGCTGAAGGAGATCATGA-3′; siMapk12_002: 5′-GCATCATGGCGGAGATGAT-3′; siTGFβ_001: 5′-CCAAGGGCTACCATGCCAA-3′; siTGFβ_002: 5′-ACCAACTATTGCTTCAGCT-3′. p38γ (WT, K56R, D179A) and GFP-LC3 expression vectors were constructed as described previously (Zhang et al. 2023, 2022).

Immunofluorescence staining

The cells were fixed with paraformaldehyde (20 min) and treated with TritonX-100 (0.2%) for 15 min. After blocking with bovine serum albumin (BSA) for half an hour, the diluted primary antibody was added and incubated at 4 °C overnight. The next day, the cells were washed with PBS, incubated with diluted secondary antibodies (Alexa Fluor 488 or 594 labeled) for 90 min, and then stained with DAPI at room temperature. Finally, immunofluorescence detection was performed using a fluorescence microscope (Eclipse 80i, Nikon, Japan).

Western blot

The experimental cells were washed twice with PBS, and the cell lysate was added for lysis. At the same time, a mixture of protease inhibitors and phosphatase inhibitors was added to prevent protein degradation. After full lysis, the cells were scraped clean and transferred to the EP tube, centrifuged at 4° C for 30 min, and the supernatant was taken for sample preparation. The appropriate concentration of SDS-PAGE was selected to separate the protein sample (20–40 μg), and the protein was transferred to the PVDF membrane using the transfer solution (Millipore, USA). After the transfer, the membrane was blocked with a rapid blocking solution, and the membrane was incubated with the diluted primary antibody overnight (4 °C). The secondary antibody needs to be selected from goat anti-rabbit or anti-mouse IgG HRP (Thermo Fisher, USA) according to different primary antibodies. After sufficient incubation, the ECL detection kit is used for color development and exposure. For the collected images, ImageJ software was used for processing and statistical analysis.

In vivo tumor xenografts in nude mice

All mouse experiments were conducted in accordance with the guidelines for the use and care of institutional animals, and all procedures were approved by the Animal Ethics Committee of West China Hospital of Sichuan University. BEL-7404 cells (5 × 106) were resuspended in 100 μL PBS and subcutaneously injected into 5–6-week-old female BALB/c nude mice (Beijing HFK Bioscience Co, Ltd.). The tumor volume was measured using an electronic caliper, and the calculation formula was as follows: volume (cm3) = L × W2 × 0.5, where L and W represent length and width. After 10 days of implantation, mice with a tumor size of 100 ~ 180 mm3 were divided into 4 groups (n = 6). When the tumor volume reached 100 mm3, the mice were orally administered with an equal volume of normal saline (DMSO control), Sora (20 mg/kg/3 days), pirfenidone (150 mg/kg/day), and the combination of Sora and PFD. After that, the long diameter and short diameter of the tumor were recorded every three days. When the maximum diameter of the transplanted tumor in the control group exceeded 1500 mm3, the administration was stopped. After all the nude mice were sacrificed, the transplanted tumor tissue was separated and weighed and photographed.

Ualcan

UALCAN (http://ualcan.path.uab.EdU) is an online analysis tool that mainly analyzes transcriptome data and clinical data of 31 cancer types in the TCGA database. We used this database to analyze the expression of MAPK family (p38α, p38β, p38γ, p38δ) in hepatocellular carcinoma and its correlation with patient prognosis.

Statistical analysis

The pictures of this study were drawn using GraphPad Prism software, and the experimental results were expressed as mean ± standard deviation (M ± SD). All the data of this study were statistically analyzed by SPSS, t test was used to compare the differences between the two groups, log-rank test was used to analyze the survival differences, and one-way analysis of variance was used to analyze the data between multiple groups. n represents the number of independent repetitions of the experiment. If not specified, repeat at least 3 times independently. The test level was α = 0.05, P < 0.05 was considered statistically significant.

Results

P38γ/MAPK12 is a targeted driving factor for the development of hepatocellular carcinoma

Through the retrieval and analysis of the existing data in the Ualcan database website, we found that the mRNA level of p38γ synthetic gene in liver cancer tissues was higher than that in normal tissues, and the survival time of patients with high expression of p38γ was significantly different from that of patients with medium/low expression (Fig. 1 E, F). After analyzing the data of p38α (Fig. 1 A, B), p38β (Fig. 1 C, D) and p38δ (Fig. 1 G, H), it was found that although the mRNA levels of these synthetic genes related to these protein kinases were different, they were not related to the prognosis of patients. This suggests that p38γ may have the potential to become a targeted therapeutic site for hepatocellular carcinoma (HCC). Therefore, we first selected four different liver cancer cell lines Huh-7, PLC/PRF/5, Hep-G2, BEL-7404 and mouse normal liver cell line AML-12, and detected the background expression of p38γ in the cells by Western Blot (Fig. 1 I). It was found that p38γ was highly expressed in hepatocellular carcinoma cells compared with normal hepatocyte AML-12, which further confirmed that p38γ was a potential targeted therapeutic site.

Fig. 1.

Fig. 1

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P38γ/MAPK12 is a targeted driving factor for the development of hepatocellular carcinoma. The expression level of p38γ in tumor tissues is related to the prognosis of patients and p38γ washighly expressed in hepatocellular carcinoma cell lines. The data of p38α (A, B), p38β (C, D), p38γ (E, F) and p38δ (G, H) in the Ualcan database website were analyzed. I, J: Western Blot was used to detect the expression of p38γ in normal liver cell line AML-12 and liver cancer cell lines Huh-7, PLC/PRF/5, Hep-G2 and BEL-7404. [UALCAN (http://ualcan.path.uab.EdU) is an online analysis tool in the TCGA database. We used this database to analyze the expression of MAPK family (p38α, p38β, p38γ, p38δ) in hepatocellular carcinoma and its correlation with patient prognosis]

P38γ can regulate the inhibitory effect of Sora on the proliferation of hepatocellular carcinoma cells

Next, we explored whether p38γhas a regulatory effect on the function of Sora. We first enriched p38γ protein by Co-Immunoprecipitation (Co-IP), and then detected the phosphorylation level of p38γ by p-p38 antibody. It was found that the phosphorylation level of p38γ in hepatocellular carcinoma cells Huh-7 and BEL-7404 did not change significantly after Sora treatment (Supplementary Fig.S1). these results suggest that there is a background activation of p38γ, which further confirms that p38γ can be used as an anticancer target for the treatment of tumor diseases. To test the effect of p38γ on the efficacy of Sora, we first used small interfering RNA to knock down the expression level of p38γ in liver cancer cells BEL-7404 and Huh-7, and then treated the cells with Sora. The results showed that knockdown of p38γ expression could enhance the inhibitory effect of Sora on the proliferation of hepatocellular carcinoma cells compared with the control cells transfected with si-NC (Fig. 2 A-F). Consistently, Doramapimod (BIRB796), an inhibitor of p38γ/δ significantly enhance the killing effect of Sora on tumor cells (Supplementary Fig.S2). In addition, we also constructed the expression plasmids of different mutants of p38γ to further confirm our experimental results, overexpressing wild-type p38γ (WT), kinase inactivated mutant p38γ (K56R) and kinase activity continuously activated mutant p38γ (D179A), respectively. The results showed that overexpression of p38γ (D179A) significantly reduced the sensitivity of HCC to Sora compared with overexpression of p38γ (K56R), indicating that the effect of p38γ on the anti-HCC effect of Sora is dependent on its protein kinase activity. Taken together, these results indicate that activated p38γ promotes the drug resistance of HCC to Sora.

Fig. 2.

Fig. 2

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P38γ can regulate the inhibitory effect of Sora on the proliferation of hepatocellular carcinoma cells. A-F: Knockdown of p38γ can enhance the inhibitory effect of Sora on the proliferation of hepatocellular carcinoma cells. Control small interfering RNA si-NC and si-p38γ were transfected into hepatocellular carcinoma cell lines BEL-7404 and Huh-7, respectively. After 24 hours of culture, different concentrations of Sora (0/2.5/5/10 μM) were added and cultured for 48 hours. CCK-8 was used to detect cell survival and determine its proliferation activity. the expression level of p38γ was detected by western blot. G-L: The effect of p38γ on the anti-HCC effect of Sora is dependent on its protein kinase activity. Vector, p38γ (WT), p38γ (K56R) and p38γ (D179A) plasmids were transfected into HCC cell lines PLC/PRF/5, Hep-G2 and Huh-7, respectively. After 24 hours, different concentrations of Sora (0/20 μM) were added and cultured for 48 hours. CCK-8 was used to detect cell survival and determine its proliferation activity. the expression level of FLAG-p38γ was detected by western blot. (n= 3, *P<0.05). **P<0.01; ***P<0.001)

PFD synergistically enhanced the inhibitory effect of sora on hepatocellular carcinoma

Since PFD was found a specific inhibitor to p38γ and showed good clinical safety, we next investigated the possible implication of Sora combined with PFD to suppress the growth of HCC. We first used different concentration gradients of Sora and pirfenidone in three liver cancer cell lines BEL-7404, Huh-7, PLC/PRF/5, and found that PFD can enhance the killing effect of Sora on liver cancer cells in vitro, and inhibit the proliferation of different liver cancer cell lines (Fig. 3 A-C) and colony formation (Fig. 3 D-I).

Fig. 3.

Fig. 3

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PFD synergistically enhanced the inhibitory effect of Sora on hepatocellular carcinoma. carcinoma. A-C: PFD enhances the killing effect of Sora on liver cancer cells. BEL-7404, Huh-7 and PLC/PRF/5 cells were treated with different concentrations of Sora (2.5/5/10/20 μM) and PFD (1/2/4 mM). After 48 h of culture, CCK-8 was used to detect cell survival and determine its proliferation activity. D-F: PFD combined with Sora inhibited the colony formation of liver cancer cells. BEL-7404, Huh-7 and PLC/PRF/5 were treated with DMSO, Sora (1.25/2.5/5μM), PFD (1/2mM) and their combination, respectively. After 14 days of culture, the colony formation assay was performed and the clone area was calculated using ImageJ software to detect the ability of cells to form clones. J-L: SynergyFinder analysis showed that Sora and PFD had a synergistic effect. BEL-7404, Huh-7 and PLC/PRF/5 cells were treated with different concentrations of Sora (2.5/5/10/20 μM) and PFD (1/2/4 mM). After 48 h of culture, CCK-8 was used to detect cell survival and determine its proliferation activity. After inputting the CCK-8 results into SynergyFinder, the Bliss synergy scores of different liver cancer cell lines were obtained. (n= 3, *P<0.05). **P<0.01; ***P<0.001)

If two different drugs are used in combination, the effect of the drug is greater than or more than the simple sum of the effects of the two drugs alone. We call these two drugs synergistic. There are many ways to analyze and judge the interaction forms of the two drugs. Here we choose SynergyFinder (https://synergyfinder.fimm.fi), a computer online analysis platform first released in 2017 and updated in 2020, for calculation and analysis. The CCK-8 results obtained from the experiment were input into SynergyFinder to obtain the Dose–response matrix and Bliss synergy score (Fig. 3 J-L). When the dose of Sora and PFD increased, the combined effect of the two drugs was significantly higher than that of the single drug. The scores of BEL-7404, Huh-7 and PLC/PRF/5 were 13.51,13.705 and 15.019, respectively, which were all higher than 10, indicating that there was a drug synergy between Sora and pirfenidone in the four liver cancer cell lines.

PFD was originally found to be an inhibitor of the TGF pathway, so we examined whether pirfenidone sensitizes Sora by acting on the TGF pathway. SRI-011381 is an agonist of TGF-β signaling pathway, while A83-01 is an inhibitor of TGF-β type I receptor. The results showed that Huh-7 liver cancer cell line and Hela human cervical cancer cell line were treated with different concentrations of two drugs combined with 5 μM Sora, respectively. It was found that there was no significant change in the effect of Sora on inhibiting cell proliferation, whether it was activating or inhibiting TGF-β pathway. This indicates that the TGF-β pathway is not involved in regulating the killing effect of Sora on tumor cells, and suggests that the synergistic killing effect of pirfenidone and Sora is not achieved by targeting the TGF-β pathway (Supplementary Fig.S3). In addition, according to the above, the expression level of p38γ in AML-12 cells is extremely low. We used different concentrations of Sora and PFD in mouse normal liver cell line AML-12, and found that in normal liver cells, the combination of Sora and PFD had no synergistic effect, and cell proliferation was not significantly inhibited (Supplementary Fig.S4). In addition, we also observed that knockdown of p38γ could abolish the sensitizing effect of PFD on Sora (Supplementary Fig.S5). In addition, we preliminarily explored the effects of p38γ and PFD on Sora resistance in HCC cells. We found that in Sora-resistant PLC/PRF-5 cells, knockdown of p38γ and PFD treatment could sensitize the response of drug-resistant cells to Sora (Supplementary Fig.S6). Taken together, these results suggest that pirfenidone may enhance the inhibitory effect of Sora on hepatocellular carcinoma through the p38γ pathway.

Inhibiting autophagy sensitizes sora

Our preliminary study found that different autophagy inhibitors can reduce the survival rate of liver cancer cells after combination therapy. Therefore, we propose a hypothesis that pirfenidone may cooperate with Sora to kill liver cancer cells by inhibiting autophagy.

We first used Sora in combination with different autophagy inhibitors, Wortmannin, SBI-0206965 and Bafilomycin A1, to treat HCC cell lines BEL-7404, PLC/PRF/5, Hep-G2, and Huh-7. The results showed that three different autophagy inhibitors can significantly enhance the killing effect of Sora on liver cancer cells (Fig. 4 A-I). Furthermore, we constructed a PLC/PRF/5 autophagy-deficient cell line by knocking out ATG5, an important regulator of autophagosome formation, using CRISPR/Cas9 system. After treating normal PLC/PRF/5 (ATG5+/+) cells and PLC/PRF/5 (ATG5−/−) cells with different concentrations of Sora, it was found that knockout of ATG5 significantly enhanced the inhibitory effect of Sora on the proliferation of PLC/PRF/5 cells (Fig. 4 J, K). Collectively, these results suggest that autophagy process is the main regulatory pathway for HCC cells to resist Sora treatment.

Fig. 4.

Fig. 4

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Inhibiting Autophagy Sensitizes Sora. BEL-7404, PLC/PRF/5, Hep-G2 and Huh-7 cells were treated with different concentrations (A-C) Wortmannin (0.25/0.5/1 μM), (D-F) SBI-0206965 (0.25/0.5/1 μM) or (G-I) Bafilomycin A1 (2.5/5/10 nM) in combination with Sora (10 uM). After 48 hours of culture, CCK-8 was used to detect cell survival and determine its proliferation activity. J-K: Knockdown of ATG5 can enhance the inhibitory effect of Sora on the proliferation of hepatocellular carcinoma cells. PLC/PRF/5-cells with ATG5 knockout were constructed, and the knockout effect was detected by Western Blot. Normal PLC/PRF/5 cells (ATG5+/+) and PLC/PRF/5 (ATG5-/-) cells were treated with different concentrations of Sora (0/5/10/20 μM). After 48 hours of culture, CCK-8 was used to detect cell survival and determine its proliferation activity. (n= 3, *P<0.05). **P<0.01; ***P<0.001)

p38γ regulates autophagy in HCC cells

Next, we examined whether p38γ could regulate the autophagy flux in HCC cells.

We used small interfering RNA to silence p38γ in three different liver cancer cell lines PLC/PRF/5, Huh-7, and BEL-7404, and then treated them with Bafilomycin A1, which can block the degradation of autophagosome. The results showed that when the expression level of p38γ was knocked down, the expression level of LC3B-II, an autophagosome marker, in the cells decreased and the expression level of p62, a substrate of autophagy, increased (Fig. 5). Taken together, these results indicate that p38γ is a driven factor for autophagy in HCC cells (Fig. 6 H).

Fig. 5.

Fig. 5

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p38γ Regulates Autophagy in HCC Cells. Knockdown of p38γ can reduce the level of autophagy in cells. The control small interfering RNA si-NC and si-p38γ were transfected into PLC/PRF/5 (A-C), Huh-7 (D-F) and BEL-7404 (G-I), respectively. After 24 hours of culture, the protein was extracted, and the expression levels of LC3B-II and p62 in the cells were detected by Western Blot, and the expression level of p38γ was detected to verify the knockout efficiency. The gray value of WB results was statistically analyzed using ImageJ software. (n= 3, *P<0.05; **P<0.01; ***P<0.001)

Fig. 6.

Fig. 6

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PFD combined with Sora can down-regulate the level of autophagy in HCC cells. A-D: PFD combined with Sora can down-regulate the level of autophagy in HCC cells. Western Blot was used to detect the expression of LC3B-II in four hepatocellular carcinoma cell lines BEL-7404, PLC/PRF/5 and Huh-7 after 48 hours of culture in the control group, Sora (10 μM) single drug group, pirfenidone (1 mM) single drug group and combined drug group. At the same time, the gray value of WB results was statistically analyzed by ImageJ software. E-G: Thecombination of Sora and pirfenidone reduced the expression level of Beclin-1 in hepatocellular carcinoma cells. Western Blot was used to detect the expression of Beclin-1 in three hepatocellular carcinoma cell lines BEL-7404 and PLC/PRF/5 after 48 hours of culture in the control group, Sora (10 μM) single drug group, pirfenidone (1 mM) single drug group andcombined drug group. At the same time, the gray value of WB results was statistically analyzed by ImageJ software. H: PFD reduces the number of autophagosomes produced. Huh-7 cells were transfected with GFP-LC3 plasmid and cultured for 24 hours, then treated with Sora (0/5/10 μM), pirfenidone (0/1 mM) and combination for 48 hours. After the cells were fixed, the formation of autophagosomes showing green fluorescence was observed under a fluorescence microscope. At the same time, the autophagosomes were counted, and the results were statistically analyzed. (n=3, *P<0.05, **P<0.01; ***P<0.001)

PFD combined with sora can down-regulate the level of autophagy in HCC cells

Since knockdown of p38γ could inhibit the autophagy, we next examined whether pirfenidone sensitizes the inhibition of Sora on HCC cells by inhibiting autophagy. We used Western Blot to detect the expression of LC3B-II in hepatocellular carcinoma cell lines BEL-7404, PLC/PRF/5 and Huh-7 cells in the control group, Sora single drug group, pirfenidone single drug group and combined drug group. The results showed that the expression level of LC3B-II was significantly decreased in hepatocellular carcinoma cells after combined treatment with pirfenidone compared with Sora alone (Fig. 6). This indicates that pirfenidone combined with Sora can down-regulate the level of autophagy in HCC. Additionally, we also found that combination of pirfenidone and Sora significantly decreased the protein level of Beclin-1, an important factor required for autophagosome formation. Consistent with the biochemical result, immunostaining result also showed that PFD reduced the number of autophagosome (Fig. 6 H). Collectively, these results indicate that PFD could inhibit the autophagy process in HCC after Sora treatment.

In vivo experiments confirmed the synergistic anti-tumor effect of PFD and Sora

Next, we investigated the synergistic anti-tumor effect of pirfenidone and Sora in BEL-7404 tumor xenografts. In vivo experiments confirmed that the combination of Sora and PFD could synergistically inhibit the growth of subcutaneous transplanted tumors in nude mice and significantly reduce the volume (Fig. 7 A, B) and weight (Fig. 7 C) of transplanted tumors. However, with the prolongation of the administration time, in the later stage of the administration stage, it can be seen that the combination group had significant cumulative toxicity, and the weight loss of mice was more obvious (Fig. 7 D), suggesting further in-depth studies are warranted before clinical application. The combination has certain toxic and side effects, and more research is needed to determine the strategy (especially the dose) and safety of the combination before clinical application.

Fig. 7.

Fig. 7

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In vivo experiments confirmed the synergistic anti-tumor effect of PFD and Sora. PFD and Sora synergistically inhibited the growth of mouse liver cancer xenografts. After the administration was stopped, the transplanted tumor tissue was isolated and weighed, and photographed (A). The line chart (B) showed the volume change of subcutaneous transplanted tumor in nude mice after administration, and the scatter plot (C) showed the end point of administration time and the weight of subcutaneous transplanted tumor in nude mice. The weight changes of mice during administration were statistically analyzed and plotted (D). Statistical methods: single factor analysis, *P<0.05; **P<0.01; ***P<0.001

In conclusion, PFD is an inhibitor of p38γ, which may affect the Beclin pathway by inhibiting the function of p38γ, thereby inhibiting the level of autophagy, and ultimately achieving the effect of sensitizing Sora.

Discussion

Drug synergy between PFD and Sora

Pirfenidone is a pyridine oral small molecule drug that has been used in clinical and preclinical studies in recent years. Because PFD has anti-fibrosis and anti-inflammatory effects, it is often used to treat pulmonary fibrosis and related diseases. Recently, more and more studies have shown that PFD has a certain anti-tumor effect. It has been observed that the probability of lung cancer in patients with pulmonary fibrosis (IPF) treated with PFD will be significantly reduced (Miura et al., 2018). In vivo studies can also confirm this conclusion. In these studies, whether PFD is combined with cisplatin, it can be clearly observed that it significantly reduces the tumorigenesis of lung cancer and pancreatic cancer and limits the growth of cancer cells, which may be achieved by changing the tumor microenvironment (Kozono et al., 2013; Mediavilla-Varela et al., 2016). In addition to these tumors mentioned above, PFD has also been confirmed in many studies to inhibit the growth and proliferation of various tumor cells, such as leiomyoma and glioma, demonstrating its anticancer potential (Kozono et al., 2013; Lee et al., 1998; Burghardt et al., 2007).

Our study also confirmed this result. Experiments in different liver cancer cell lines found that different concentrations of pirfenidone can enhance the killing effect of Sora on liver cancer cells in vitro, and inhibit the proliferation activity and colony formation ability of liver cancer cells. High concentrations of pirfenidone may also have a certain inhibitory effect on tumor cell proliferation. SynergyFinder online analysis software was used to analyze the results, and it was found that there was indeed a drug synergy between Sora and pirfenidone. In addition, in vivo experiments, we also confirmed that the combined use of Sora and pirfenidone can synergistically inhibit the growth of subcutaneous hepatocellular carcinoma xenografts in nude mice, significantly reducing the volume and weight of xenografts. These experimental results have proved that pirfenidone can inhibit tumor cell proliferation and synergistic Sora cell killing effect.

PFD may cooperate with Sora through a variety of ways. Studies have confirmed that pirfenidone can maintain the stability of the tumor microenvironment and increase the route of chemotherapy drugs into the tumor by reducing the levels of hyaluronic acid and collagen in ECM (Polydorou et al., 2017). In addition, pirfenidone can also directly target CEMIP (Cell Migration Inducing Hyaluronidase 1) and inhibit the CEMIP/TGF-β1/Smad signaling pathway. The combination of Sora and pirfenidone can reduce the lung metastasis of hepatocellular carcinoma and has good anti-tumor activity(Liu et al., 2023). It is worth noting that according to our findings, with the prolongation of administration time, the body weight of mice will be reduced to varying degrees, suggesting that the combination has certain toxic and side effects, and more research is needed before clinical application to determine the strategy (especially dose) and safety of the combination.

At the same time, pirfenidone has a wide range of anti-fibrosis effects. Recent studies have revealed that pirfenidone is a specific inhibitor of p38γ, and the p38 family is a cascade kinase involved in controlling cell responses to cytokines and stress. In addition to the results obtained in this study, that is, p38γ has a down-regulation of autophagy levels, there are also studies showing that tumor-associated neutrophils (TANs) recruit macrophages and Treg cells to hepatocellular carcinoma by expressing chemokine ligands 2 and 17 (CCL2 and CCL17), thereby promoting tumor growth and Sora resistance. Inhibition of AKT and p38 can block the expression of CCL2 and CCL17 in TANs and enhance the efficacy of Sora (Zhou et al., 2016). However, whether p38γ is involved in the regulation of this process needs further study to reveal.

Cells may increase resistance to Sora by up-regulating autophagy levels

Autophagy is a spontaneous physiological process of cells and a mechanism for cell self-clearance. It is regulated by autophagy-related genes (ATG), so that intracellular proteins or damaged organelles are broken down by lysosomes. Although it is generally believed that enhanced autophagy activity increases cell death and inhibits cell proliferation, the autophagy process of cancer cells is actually complex. In some cases, increased levels of autophagy may instead support the survival of cancer cells. (White and Dipaola 2009; Shintani and Klionsky 2004). Different from other ways of death, in the physiological state, the cell itself has a background autophagy process, also known as basic autophagy, which is a normal physiological phenomenon. When it is stimulated by other pathological stimuli, induced autophagy occurs. The initiation of autophagy can enhance the viability of tumor cells when they face external pressure. This process is one of the important factors that promote cell resistance to Sora (Thorburn 2014).

In order to explore the combined sensitizing effect of Pirfenidone on Sorafenib, we used different experimental methods to systematically screen a variety of programmed cell death methods. It has been found that autophagy may be an indispensable part of it. The results showed that the use of autophagy inhibitors such as Wortmannin, SBI-0206965 or Bafilomycin A1 could significantly enhance the killing effect of Sora on HCC cells. In addition, knockdown of ATG5 also significantly enhanced the inhibitory effect of Sora on the proliferation of PLC / PRF / 5 cells, suggesting that reducing the level of autophagy may be an effective strategy to improve the sensitivity of tumor cells to Sora. Studies (Luo et al., 2016) have confirmed that in patients with high expression of ATG7, due to the activation of autophagy and the increase of intracellular autophagy level, these patients have a poor prognosis and resistance to Sora treatment. Based on these results, we can affirm a molecular mechanism of low sensitivity or drug resistance of tumor cells to Sora, that is, cells increase resistance to Sora by up-regulating autophagy level. The increase of autophagy level within a certain range helps to protect tumor cells from external survival pressure, and down-regulation of autophagy level can increase the killing effect of Sora on hepatocellular carcinoma.

P38γ reduced the level of autophagy in hepatocellular carcinoma cells

In this study, we found that after knocking down the expression level of p38γ by si-p38γ, the expression level of LC3B-II in PLC/PRF/5, Huh-7 and BEL-7404 cells decreased, and the expression level of p62 increased, indicating that down-regulating the expression level of p38γ can inhibit the autophagy process. This conclusion is exciting. Previous studies on the relationship between p38 MAPK pathway and autophagy often focused on p38α/β, while the regulation of p38γ in cells is very limited. For example, the results of Henson et al. (Henson et al, 2014) confirmed that blocking p38 MAPK in senescent CD1 + T lymphocytes can induce autophagy in a mTOR-independent manner by enhancing the interaction between p38 interacting protein (p38IP) and autophagy protein 9 (ATG9). On the contrary, autophagy can also promote cell survival by inhibiting p62-mediated p38 MAPK pathway activation (Qiang et al., 2013). The above research results suggest that inhibiting the activation of p38α may activate autophagy, and our results suggest that inhibiting p38γ can down-regulate the level of autophagy. This conclusion suggests that in the autophagy regulatory network, the regulatory functions of p38α and p38γ may be different, which is also one of the innovations of this study.

Beclin1 (BECN1) is a homologous gene of yeast autophagy gene Atg6/Vps30 in mammals, and it is also a specific gene involved in autophagy in mammals. The close relationship between Beclin1 and tumors plays an important role in the occurrence and development of tumors, and is an important link between autophagy and tumors. Here, we found that after blocking the autophagy flow with Bafilomycin A1, compared with Sora alone, the combined use of p38γ inhibitor pirfenidone in hepatocellular carcinoma cells BEL-7404, PLC/PRF/5, and Hep-G2 could significantly reduce the expression level of Beclin-1 implying that p38γ may inhibit autophagy by down-regulating the expression of Beclin-1. Future studies are warranted to address these possibilities.

P38γ is a potential new target for the treatment of liver cancer

As mentioned above, through a series of experiments, it can be seen that after transfection of si-p38γ and si-NC, reducing the expression of p38γ significantly enhanced the inhibitory effect of Sorafenib on HCC, which may be caused by reducing the level of autophagy. In addition, our other research evidence supports that p38γ is a very promising new target for the treatment of liver cancer. We observed that the level of p-p38γ in tumor cells such as liver cancer increased, indicating that p38γ was activated. Patients with different expression levels of P38γ have significant differences in their survival time, which can also be found in HCC tissues, that is, the mRNA level of p38γ synthesis gene is higher than that of normal tissues.

P38γ can be activated when the external environmental pressure changes, or under the action of mitotic signals. When p38γ is activated, it can further promote the occurrence and development of cancer.(Qi et al., 2011). For example, overexpression of p38γ can promote the progression of osteosarcoma (Shi et al., 2020). Previous studies have confirmed that ACAT2 and COL1 A are common fibrosis markers in the early stage of liver cancer, which may be related to their encoding actin and collagen, respectively. The expression of p38γ is directly related to their expression, which indirectly confirms the potential of p38γ as a new target for the treatment of hepatocellular carcinoma. When p38γ is increased, liver cancer is more likely to progress (Fu et al., 2011). Cyclin-dependent kinase (CDK) has substrate specificity and inhibitory sensitivity, as well as p38γ. When the level of CDK-cyclin complex is low, p38γ can activate the non-classical CDK pathway in a cyclin-independent manner, thereby initiating the cell cycle and protecting tumor cells from physical and chemical stimuli and damage (Xu et al., 2021). Taken together, these evidences clearly demonstrate the importance of p38γ in the occurrence and development of hepatocellular carcinoma and support its potential as a new target for future treatment of hepatocellular carcinoma.

In summary, our study provides new ideas and treatment strategies for the anti-tumor effect of pirfenidone, solving the problem of drug resistance in the practical clinical application of Sora, and further optimizing the treatment of patients with advanced liver cancer.

Supplementary Information

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Authors' contributions

FB. and CLZ conceived the project. CLZ and CH designed the experiments. CH performed the majority of the experiments and analyzed the data. JJL, YCD and QT helped with data collection and analysis. CH and CLZ wrote the manuscript. FB supervised the study.

Funding

This research was supported by the National Natural Science Foundation of China (grant numbers: 82073059, 32000533); 1.3.5 Project for Disciplines of excellence, West China Hospital, Sichuan University (grant number: ZYGD18005).

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethical approval 

All procedures were approved by the Animal Ethics Committee of West China Hospital of Sichuan University.

Competing interests

The authors declare no competing interests.

Footnotes

Chen Huang and Chenliang Zhang contributed equally in this article.

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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