Depletion of regulatory T cells enhancing the anti-tumor effect of in situ vaccination in solid tumors.

The emergence of immune checkpoint inhibitors (ICIs) has revolutionized the clinical treatment for tumor. However, the low response rate of ICIs remains the major obstacle for curing patients and effective approaches for patients with primary or secondary resistance to ICIs remain lacking. In this study, immune stimulating agent unmethylated CG-enriched (CpG) oligodeoxynucleotide (ODN) was locally injected into the tumor to trigger a robust immune response to eradicate cancer cells, while anti-CD25 antibody was applied to remove immunosuppressive regulatory T cells, which further enhanced the host immune activity to attack tumor systematically. The combination of CpG and anti-CD25 antibody obtained notable regression in mouse melanoma model. Furthermore, rechallenge of tumor cells in the xenograft model has resulted in smaller tumor volume, which demonstrated that the combinational treatment enhanced the activity of memory T cells. Remarkably, this combinational therapy presented significant efficacy on multiple types of tumors as well and was able to prevent relapse of tumor partially. Taken together, our combinational immunotherapy provides a new avenue to enhance the clinical outcomes of patients who are insensitive or resistant to ICIs treatments.

Circular RNAs: Potential biomarkers and therapeutic targets for autoimmune diseases.

The outcomes and prognosis of autoimmune diseases depend on early diagnosis and effective treatments. However, symptoms of early autoimmune diseases are often remarkably similar to many inflammatory diseases, leading to difficulty in precise diagnosis. Circular RNAs (circRNAs) belong to a novel class of endogenous RNAs, functioning as microRNA (miRNA) sponges or participating in protein coding. It has been shown in many studies that patients with autoimmune diseases have aberrant circRNA expression in liquid biopsy samples (such as plasma, saliva, and urine). Thus, circRNAs are potential biomarkers for the diagnosis and prognosis of autoimmune diseases. Moreover, overexpression and depletion of target circRNAs can be utilized as possible therapeutic approaches for treating autoimmune diseases. In this review, we summarized recent progress in the roles of circRNAs in the pathogenesis of autoimmune diseases, including rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, and type 1 diabetes. We also discussed their potential as biomarkers and therapeutic targets.

ZYZ384 suppresses the growth of EGFR-mutant non-small cell lung cancer by activating JNK/MAPK signaling pathway.

The emergency of tyrosine kinase inhibitors has remarkably enhanced the clinical outcomes of cancer therapy, especially the use of EGFR inhibitors for non-small cell lung cancer (NSCLC). However, acquired resistance is inevitable after 8-12 months treatment. New agents or treatments are urgently required to resolve this problem. In this study, we identified that compound ZYZ384 can selectively inhibit the growth of gefitinib-resistant (G-R) lung cancer cells, without affecting that of normal lung epithelial cells. ZYZ384 induced G2 arrest in G-R NSCLC cells, decreasing the expression of Cyclin B1 and increasing the expression of P21. Meanwhile, ZYZ384 also induced apoptosis in NSCLC cells and correspondingly increased the expression of cleaved Caspase 3, 8, and 9 proteins. The expression of p-JNK, p-P38, and p-ERK were also increased in H1975 NSCLC cells treated with ZYZ384. Finally, we observed that the JNK inhibitor effectively reversed the pro-apoptotic effect of ZYZ384. In conclusion, ZYZ384 is a potential therapeutic agent to inhibit the growth of NSCLCs with EGFR mutations through activating JNK, which will help the development of related anticancer drugs.

Longitudinal high-dimensional analysis identifies immune features associating with response to anti-PD-1 immunotherapy.

Response to immunotherapy widely varies among cancer patients and identification of parameters associating with favourable outcome is of great interest. Here we show longitudinal monitoring of peripheral blood samples of non-small cell lung cancer (NSCLC) patients undergoing anti-PD1 therapy by high-dimensional cytometry by time of flight (CyTOF) and Meso Scale Discovery (MSD) multi-cytokines measurements. We find that higher proportions of circulating CD8+ and of CD8+CD101hiTIM3+ (CCT T) subsets significantly correlate with poor clinical response to immune therapy. Consistently, CD8+ T cells and CCT T cell frequencies remain low in most responders during the entire multi-cycle treatment regimen; and higher killer cell lectin-like receptor subfamily G, member 1 (KLRG1) expression in CCT T cells at baseline associates with prolonged progression free survival. Upon in vitro stimulation, CCT T cells of responders produce significantly higher levels of cytokines, including IL-1ß, IL-2, IL-8, IL-22 and MCP-1, than of non-responders. Overall, our results provide insights into the longitudinal immunological landscape underpinning favourable response to immune checkpoint blockade therapy in lung cancer patients.

Detection of Single Cancer Cell Multidrug Resistance With Single Cell Bioanalyzer.

Objectives: Despite the development of various cancer treatment methods, chemotherapy remains the most common approach for treating cancer. The risk of tumors acquiring resistance to chemotherapy remains a significant hurdle to the successful treatment of various types of cancer. Therefore, overcoming or predicting multidrug resistance in clinical treatment is essential. The detection of circulating tumor cells (CTCs) is an important component of liquid biopsy and the diagnosis of cancer. This study aims to test the feasibility of single-cell bioanalyzer (SCB) and microfluidic chip technology in identifying patients with cancer resistant to chemotherapy and propose new methods to provide clinicians with new choices. Methods: In this study, we used rapidly isolated viable CTCs from the patient blood samples method combined with SCB technology and a novel microfluidic chip, to predict whether patients with cancer are resistant to chemotherapy. SCB and microfluidic chip were used to select single CTCs, and the accumulation of chemotherapy drug was fluorescently measured in real time on these cells in the absence and presence of permeability-glycoprotein inhibitors. Results: Initially, we successfully isolated viable CTCs from the blood samples of patients. Additionally, the present study accurately predicted the response of 4 lung cancer patients to chemotherapeutic drugs. In addition, the CTCs of 17 patients with breast cancer diagnosed at Zhuhai Hospital of Traditional Chinese and Western Medicine were assessed. The results indicated that 9 patients were sensitive to chemotherapeutic drugs, 8 patients were resistant to a certain degree, and only 1 was completely resistant to chemotherapy. Conclusion: The present study indicated that the SCB technology could be used as a prognostic assay to evaluate the CTCs response to available drugs and guide physicians to treatment options that are most likely to be effective.

DMU-212 against EGFR-mutant non-small cell lung cancer via AMPK/PI3K/Erk signaling pathway.

Although some important advances have been achieved in clinical and diagnosis in the past few years, the management of non-small cell lung cancer (NSCLC) is ultimately dissatisfactory due to the low overall cure and survival rates. Epidermal growth factor (EGFR) has been recognized as a carcinogenic driver and is a crucial pharmacological target for NSCLC. DMU-212, an analog of resveratrol, has been reported to have significant inhibitory effects on several types of cancer. However, the effect of DMU-212 on lung cancer remains unclear. Therefore, this study aims to determine the effects and underlying mechanism of DMU-212 on EGFR-mutant NSCLC cells. The data found that the cytotoxicity of DMU-212 on three EGFR-mutant NSCLC cell lines was significantly higher than that of normal lung epithelial cell. Further study showed that DMU-212 can regulate the expression of cell cycle-related proteins including p21 and cyclin B1 to induce G2/M phase arrest in both H1975 and PC9 cells. Moreover, treatment with DMU-212 significantly promoted the activation of AMPK and simultaneously down-regulated the expression of EGFR and the phosphorylation of PI3K, Akt and ERK. In conclusion, our study suggested that DMU-212 inhibited the growth of NSCLCs via targeting of AMPK and EGFR.

Pharmacokinetics, absorption and transport mechanism for ginseng polysaccharides.

BACKGROUND: Ginseng polysaccharide (GP) is one of the most abundant components in Panax ginseng. However, the absorption pathways and mechanisms of GPs have not been investigated systematically due to the challenges of their detection. METHODS: The fluorescein isothiocyanate derivative (FITC) was employed to label GP and ginseng acidic polysaccharide (GAP) to obtain target samples. HPLC-MS/MS assay was used to determine the pharmacokinetics of GP and GAP in rats. The Caco-2 cell model was used to investigate the uptake and transport mechanisms of GP and GAP in rats. RESULTS: Our results demonstrated that the absorption of GAP was more than that of GP in rats after gavage administration, while there was no significant difference between both after intravenous administration. In addition, we found that GAP and GP were more distributed in the kidney, liver and genitalia, suggesting that GAP and GP are highly targeted to the liver, kidney and genitalia. Importantly, we explored the uptake mechanism of GAP and GP. GAP and GP are endocytosed into the cell via lattice proteins or niche proteins. Both are transported lysosomally mediated to the endoplasmic reticulum (ER) and then enter the nucleus through the ER, thus completing the process of intracellular uptake and transportation. CONCLUSION: Our results confirm that the uptake of GPs by small intestinal epithelial cells is primarily mediated via lattice proteins and the cytosolic cellar. The discovery of important pharmacokinetic properties and the uncovering of the absorption mechanism provide a research rationale for the research of GP formulation and clinical promotion.

ß-Elemene enhances erlotinib sensitivity through induction of ferroptosis by upregulating lncRNA H19 in EGFR-mutant non-small cell lung cancer.

Nearly half of all Asian non-small cell lung cancer (NSCLC) patients harbour epidermal growth factor receptor (EGFR) mutations, and first-generation EGFR tyrosine kinase inhibitors (TKIs) are one of the first-line treatments that have improved the outcomes of these patients. Unfortunately, 20% of these patients can not benefit from the treatment. The basis of this primary resistance is poorly understood. Therefore, overcoming EGFR-TKI primary resistance and maintaining the efficacy of TKIs has become a key issue. ß-Elemene, a sesquiterpene compound extracted from Curcuma aromatica Salisb. (wenyujing), has shown potent antitumor effects. In this research, we found that ß-elemene combined with erlotinib enhanced the cytotoxicity of erlotinib to primary EGFR-TKI-resistant NSCLC cells with EGFR mutations and that ferroptosis was involved in the antitumor effect of the combination treatment. We found that lncRNA H19 was significantly downregulated in primary EGFR-TKI-resistant NSCLC cell lines and was upregulated by the combination treatment. Overexpression or knockdown of H19 conferred sensitivity or resistance to erlotinib, respectively, in both in vitro and in vivo studies. The high level of H19 enhanced the cytotoxicity of erlotinib by inducing ferroptosis. In conclusion, our data showed that ß-elemene combined with erlotinib could enhance sensitivity to EGFR-TKIs through induction of ferroptosis via H19 in primary EGFR-TKI-resistant lung cancer, providing a promising strategy to overcome EGFR-TKI resistance in NSCLC patients.

Overcoming Suppressive Tumor Microenvironment by Vaccines in Solid Tumor.

Conventional vaccines are widely used to boost human natural ability to defend against foreign invaders, such as bacteria and viruses. Recently, therapeutic cancer vaccines attracted the most attention for anti-cancer therapy. According to the main components, it can be divided into five types: cell, DNA, RNA, peptide, and virus-based vaccines. They mainly perform through two rationales: (1) it trains the host immune system to protect itself and effectively eradicate cancer cells; (2) these vaccines expose the immune system to molecules associated with cancer that enable the immune system to recognize and destroy cancer cells. In this review, we thoroughly summarized the potential strategies and technologies for developing cancer vaccines, which may provide critical achievements for overcoming the suppressive tumor microenvironment through vaccines in solid tumors.

Holistic immunomodulation for small cell lung cancer.

Small cell lung cancer (SCLC) is characterized by a high mortality rate, rapid growth, and early metastasis, which lead to a poor prognosis. Moreover, limited clinical treatment options further lower the survival rate of patients. Therefore, novel technology and agents are urgently required to enhance clinical efficacy. In this review, from a holistic perspective, we summarized the therapeutic targets, agents and strategies with the most potential for treating SCLC, including chimeric antigen receptor (CAR) T therapy, immunomodulating antibodies, traditional Chinese medicines (TCMs), and the microbiota, which have been found recently to improve the clinical outcomes and prognosis of SCLC. Multiomics technologies can be integrated to develop effective diagnostic methods and identify new targets for new drug discovery in SCLC. We discussed in depth the feasibility, potential, and challenges of these new strategies, as well as their combinational treatments, which may provide promising alternatives for enhancing the clinical efficacy of SCLC in the future.

Tumor PKCδ instigates immune exclusion in EGFR-mutated non-small cell lung cancer.

BACKGROUND: The recruitment of a sufficient number of immune cells to induce an inflamed tumor microenvironment (TME) is a prerequisite for effective response to cancer immunotherapy. The immunological phenotypes in the TME of EGFR-mutated lung cancer were characterized as non-inflamed, for which immunotherapy is largely ineffective. METHODS: Global proteomic and phosphoproteomic data from lung cancer tissues were analyzed aiming to map proteins related to non-inflamed TME. The ex vivo and in vivo studies were carried out to evaluate the anti-tumor effect. Proteomics was applied to identify the potential target and signaling pathways. CRISPR-Cas9 was used to knock out target genes. The changes of immune cells were monitored by flow cytometry. The correlation between PKCδ and PD-L1 was verified by clinical samples. RESULTS: We proposed that PKCδ, a gatekeeper of immune homeostasis with kinase activity, is responsible for the un-inflamed phenotype in EGFR-mutated lung tumors. It promotes tumor progression by stimulating extracellular matrix (ECM) and PD-L1 expression which leads to immune exclusion and assists cancer cell escape from T cell surveillance. Ablation of PKCδ enhances the intratumoral penetration of T cells and suppresses the growth of tumors. Furthermore, blocking PKCδ significantly sensitizes the tumor to immune checkpoint blockade (ICB) therapy (αPD-1) in vitro and in vivo model. CONCLUSIONS: These findings revealed that PKCδ is a critical switch to induce inflamed tumors and consequently enhances the efficacy of ICB therapy in EGFR-mutated lung cancer. This opens a new avenue for applying immunotherapy against recalcitrant tumors.

PA-MSHA induces inflamed tumor microenvironment and sensitizes tumor to anti-PD-1 therapy.

A low response rate to immune checkpoint inhibitor (ICI) therapy has impeded its clinical use. As reported previously, an inflamed tumor microenvironment (TME) was directly correlated with patients' response to immune checkpoint blockade (ICB). Thus, restoring the cytotoxic effect of immune cells in the TME is a promising way to improve the efficacy of ICB and overcome primary resistance to immunotherapy. The effect of Pseudomonas aeruginosa mannose-sensitive-hemagglutinin (PA-MSHA) in facilitating T cell activation was determined in vitro and in vivo. Subsets of immune cells were analyzed by flow cytometry. Proteomics was carried out to comprehensively analyze the discriminated cellular kinases and transcription factors. The combinational efficacy of PA-MSHA and αPD-1 therapy was studied in vivo. In this study we demonstrated that PA-MSHA, which is a clinically used immune adjuvant, effectively induced the anti-tumor immune response and suppressed the growth of non-small cell lung cancer (NSCLC) cells. PA-MSHA showed great potential to sensitize refractory "cold" tumors to immunotherapy. It effectively enhanced macrophage M1 polarization and induced T cell activation. In vivo, in combination with αPD-1, PA-MSHA suppressed tumor growth and prolonged the survival time of allograft model mice. These results indicate that PA-MSHA is a potent agent to stimulate immune cells infiltration into the TME and consequently induces inflammation in tumors. The combination of PA-MSHA with αPD-1 is a potential strategy to enhance the clinical response rate to ICI therapy.

Down-regulating Nrf2 by tangeretin reverses multiple drug resistance to both chemotherapy and EGFR tyrosine kinase inhibitors in lung cancer.

Multiple drug resistance (MDR) is the major obstacle for both chemotherapy and molecular-targeted therapy for cancer, which is mainly caused by overexpression of ABC transporters or genetic mutation of drug targets. Based on previous studies, we hypothesized that ROS/Nrf2 is the common target for overcoming acquired drug resistance to both targeted therapy and chemotherapy treatments. In this study, we firstly proved that the levels of ROS and Nrf2 were remarkably up-regulated in both H1975 (Gefitinib-resistant lung cancer cells with T790M) and A549/T (paclitaxel-resistant) cells, which is consistent with the clinical database analysis results of lung cancer patients that Nrf2 expression level is negatively related to survival rate. Nrf2 Knockdown with siRNA or tangeretin (TG, a flavonoid isolated from citrus peels) inhibited the MDR cell growth by suppressing the Nrf2 pathway, and efficiently enhanced the anti-tumor effects of paclitaxel and AZD9291 (the third generation of TKI) in A549/T or H1975, respectively. Moreover, TG sensitized A549/T cells-derived xenografts to paclitaxel via inhibiting Nrf2 and its downstream target P-gp, leading to an increased paclitaxel concentration in tumors. Collectively, targeting Nrf2 to enhance ROS may be a common target for overcoming the acquired drug resistance and enhancing the therapeutic effects of chemotherapy and molecular-targeted therapy.

Novel clinical biomarkers in blood and pleural effusion for diagnosing patients with tuberculosis distinguishing from malignant tumor.

Pleural effusion (PE) is a common manifestation of tuberculosis (TB) and malignant tumors but tuberculous PE (TPE) is difficult to distinguish from malignant PE (MPE), especially by noninvasive detection indicators. This study aimed to find effective detection indices in blood and PE for differentiating TB from a malignant tumor. A total of 815 patients who were diagnosed with TB or cancer in Hubei Shiyan Taihe Hospital from 2014 to 2017 were collected. Amongst them, 717 were found to have PE by thoracoscopy. Clinical characteristics, patients' blood parameters and PE indicator information were summarized for analysis. Patients with MPE had higher percentages to be bloody and negative of Rivalta test in PE than those with TPE. For clinical indicators, comparison of the specific parameters in blood showed that 18 indicators were higher in the TPE group than in the MPE group. By contrast, 12 indicators were higher in the MPE group than in the TPE group (P < .01). In addition, in PE tests, 3 parameters were higher in the TPE group, whereas other 4 parameters were higher in the MPE group (P < .01). Then, for clinical diagnosing practice, ROC analysis and principal component analysis were applied. The top 6 relevant indicators with area under curve over 0.70 were screened out as follows: hydrothorax adenosine dehydrogenase (pADA, 0.90), hydrothorax high-sensitivity C reactive protein (0.79), percentage of blood monocyte (sMONp, 0.75), blood high-sensitivity C reactive protein (sHsCRP, 0.73), erythrocyte sedimentation rate (0.71) and blood D-dimer (0.70). Moreover, logistic regression model revealed that a specific combination of 3 biomarkers, namely, pADA, sMONp and sHsCRP, could enhance the distinguishment of TB from malignant tumor with PE (area under curve = 0.944, 95% confidence interval = 0.925-0.964). The diagnostic function of the top single marker pADA in patients from different groups was analyzed and it was found to maintain high specificity and sensitivity. The 6 indicators, namely, pADA, hydrothorax high-sensitivity C reactive protein, sMONp, sHsCRP, sESR and blood D-dimer, showed significant diagnostic value for clinicians. Further, the combination of pADA, sMONp and sHsCRP has high accuracy for differential diagnosis for the first time. Most interestingly, the single marker pADA maintained high specificity and sensitivity in patients with different statuses and thus has great value for rapid and accurate diagnosis of suspected cases.

Nanotechnology-based chimeric antigen receptor T-cell therapy in treating solid tumor.

Chimeric Antigen Receptor (CAR) T cells have changed the therapeutic landscape of hematological malignancies with overwhelming success. The clinical success of CAR T-cell therapy in hematologic malignancies has fueled interest in exploring the technology in solid tumors. However, the treatment of solid tumors presents a unique set of challenges compared to hematological tumors. The biggest impediments to the success of CAR T cell treatment are the paucity of tumor-specific antigens that are produced selectively and uniformly and the immunosuppressive tumor microenvironment. To overcome these significant challenges, nanotechnology has been involved to improve the efficacy of CAR-T cells. In this review, we systematically introduced the components of different generations of CARs and summarized recent innovations in nano-based CAR-T cell therapy to conquer therapeutically resistant non-hematologic malignancies, including mRNA and hydrogel-based CAR T cells delivery, photothermal-remodeling, and tumor microenvironment-based CAR T cell therapy. These nanotechnologies remarkably facilitate in vivo generation of CAR T cells and hold promise as a therapeutic platform to treat solid tumors and even other diseases.

Bacteria-based nanodrug for anticancer therapy.

Bacteria-based immunotherapy has become a promising strategy to induce innate and adaptive responses for fighting cancer. The advantages of bacteriolytic tumor therapy mainly lie in stimulation of innate immunity and colonization of some bacteria targeting the tumor microenvironment (TME). These bacteria have cytotoxic proteins and immune modulating factors that can effectively restrain tumor growth. However, cancer is a multifactorial disease and single therapy is typically unable to eradicate tumors. Rapid progress has been made in combining bacteria with nanotechnology. Using the nanomolecular properties of bacterial products for tumor treatment preserves many features from the original bacteria while providing some unique advantages. Nano-bacterial therapy can enhance permeability and retention of drugs, increase the tolerability of the targeted drugs, promote the release of immune cell mediators, and induce immunogenic cell death pathways. In addition, combining nano-bacterial mediated antitumor therapeutic systems with modern therapy is an effective strategy for overcoming existing barriers in antitumor treatment and can achieve satisfactory therapeutic efficacy. Overall, exploring the immune antitumor characteristics of adjuvant clinical treatment with bacteria can provide potential efficacious treatment strategies for combatting cancer.

Decoding Lung Cancer at Single-Cell Level.

Lung cancer is the leading cause of cancer death due to its high degree of malignancy, rapid growth, and early metastasis. Recent studies have found that lung cancer has a high degree of heterogeneity which is characterized by the mixture of different tumor cell types. However, the driving genetic/epigenetic mechanism of lung cancer heterogeneity, how different types of cells interact, and the relationship between heterogeneity and drug resistance have been poorly understood. Single-cell technology can decompose high throughput sequencing information into each cell and provide single-cell information in high resolution. By using single-cell analysis, researchers can not only fully understand the molecular characteristics of different cell types in the same tissue, but also define completely new cell types. Thus, single-cell analysis has been widely utilized in systems biology, drug discovery, disease diagnosis and precision medicine. We review recent exploration of the mechanism of heterogeneity, tumor microenvironment and drug resistance in lung cancer by using single-cell analysis. We propose that the recent findings may pave new ways for the treatment strategies of lung cancer.

Chelerythrine ameliorates rheumatoid arthritis by modulating the AMPK/mTOR/ULK-1 signaling pathway.

BACKGROUND: Rheumatoid arthritis (RA) is a long-term, progressive, and disabling autoimmune disease. It causes inflammation, swelling and pain in and around the joints and other body organs. Currently, no cure is available for RA. Clinical interventions can only relieve the condition, and at least 30% of RA patients do not respond to first­line therapy. This means that the development of more effective therapies against RA is urgently needed. OBJECTIVE: This study aimed to assess the anti-rheumatoid arthritis effect of chelerythrine (CLT) and explore its mechanism of action. METHODS: The cytotoxic effect of CLT on human rheumatoid arthritis fibroblast-like synoviocyte (HFLS-RA) cells and HFLS-normal cells were measured by MTT assay. The growth and migration of HFLS-RA cells were determined by colony-formation and wound-healing assay. The level of intracellular reactive oxygen species (ROS) was detected using the DCFH-DA reagent. Cell apoptosis was measured by flow cytometry, TUNEL staining, caspase 3 activity, as well as the activation of apoptosis related proteins. In addition, the levels of autophagy related markers such as LC3B and P62 were determined by immunocytochemistry and western blotting. Lastly, the anti-RA effect of CLT was evaluated in an Adjuvant-Induced Arthritis(AIA) rat model and the severity of arthritis was detected and quantified using macroscopic inspection and X­ray imaging. RESULTS: We discovered that treatment with CLT effectively inhibited the migration and colony-formation of the HFLS-RA cells and resulted in cell death. Moreover, CLT increased the intracellular level of ROS and the apoptotic rate of HFLS-RA by activating the AMPK/mTOR/ULK-1 signaling pathways. In vivo study showed CLT effectively ameliorated AIA in rats, protecting them from inflammation and bone damage. CONCLUSION: Our study shows CLT is an effective agent for ameliorating RA in vitro and in vivo by modulation of the AMPK/mTOR/ULK-1 signaling pathway. These findings indicate that CLT is a great potential candidate for development as a therapeutic agent for the prevention and treatment of RA.

Xihuang pills induce apoptosis in hepatocellular carcinoma by suppressing phosphoinositide 3-kinase/protein kinase-B/mechanistic target of rapamycin pathway.

BACKGROUND: The phosphoinositide 3-kinase/protein kinase-B/mechanistic target of rapamycin (PI3K/Akt/mTOR) signalling pathway is crucial for cell survival, differentiation, apoptosis and metabolism. Xihuang pills (XHP) are a traditional Chinese preparation with antitumour properties. They inhibit the growth of breast cancer, glioma, and other tumours by regulating the PI3K/Akt/mTOR signalling pathway. However, the effects and mechanisms of action of XHP in hepatocellular carcinoma (HCC) remain unclear. Regulation of the PI3K/Akt/mTOR signalling pathway effectively inhibits the progression of HCC. However, no study has focused on the XHP-associated PI3K/Akt/mTOR signalling pathway. Therefore, we hypothesized that XHP might play a role in inhibiting HCC through the PI3K/Akt/mTOR signalling pathway. AIM: To confirm the effect of XHP on HCC and the possible mechanisms involved. METHODS: The chemical constituents and active components of XHP were analysed using ultra-performance liquid chromatography-quadrupole time of flight mass spectrometry (UPLC-Q-TOF-MS). Cell-based experiments and in vivo xenograft tumour experiments were utilized to evaluate the effect of XHP on HCC tumorigenesis. First, SMMC-7721 cells were incubated with different concentrations of XHP (0, 0.3125, 0.625, 1.25, and 2.5 mg/mL) for 12 h, 24 h and 48 h. Cell viability was assessed using the CCK-8 assay, followed by an assessment of cell migration using a wound healing assay. Second, the effect of XHP on the apoptosis of SMMC-7721 cells was evaluated. SMMC-7721 cells were stained with fluorescein isothiocyanate and annexin V/propidium iodide. The number of apoptotic cells and cell cycle distribution were measured using flow cytometry. The cleaved protein and mRNA expression levels of caspase-3 and caspase-9 were detected using Western blotting and quantitative reverse-transcription polymerase chain reaction (RT-qPCR), respectively. Third, Western blotting and RT-qPCR were performed to confirm the effects of XHP on the protein and mRNA expression of components of the PI3K/Akt/mTOR signalling pathway. Finally, the effects of XHP on the tumorigenesis of subcutaneous hepatocellular tumours in nude mice were assessed. RESULTS: The following 12 compounds were identified in XHP using high-resolution mass spectrometry: Valine, 4-gingerol, myrrhone, ricinoleic acid, glycocholic acid, curzerenone, 11-keto-ß-boswellic acid, oleic acid, germacrone, 3-acetyl-9,11-dehydro-ß-boswellic acid, 5ß-androstane-3,17-dione, and 3-acetyl-11-keto-ß-boswellic acid. The cell viability assay results showed that treatment with 0.625 mg/mL XHP extract decreased HCC cell viability after 12 h, and the effects were dose- and time-dependent. The results of the cell scratch assay showed that the migration of HCC cells was significantly inhibited in a time-dependent manner by the administration of XHP extract (0.625 mg/mL). Moreover, XHP significantly inhibited cell migration and resulted in cell cycle arrest and apoptosis. Furthermore, XHP downregulated the PI3K/Akt/mTOR signalling pathway, which activated apoptosis executioner proteins (e.g., caspase-9 and caspase-3). The inhibitory effects of XHP on HCC cell growth were determined in vivo by analysing the tumour xenograft volumes and weights. CONCLUSION: XHP inhibited HCC cell growth and migration by stimulating apoptosis via the downregulation of the PI3K/Akt/mTOR signalling pathway, followed by the activation of caspase-9 and caspase-3. Our findings clarified that the antitumour effects of XHP on HCC cells are mediated by the PI3K/Akt/mTOR signalling pathway, revealing that XHP may be a potential complementary therapy for HCC.