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Background: Apoptosis resistance of hepatocellular carcinoma (HCC) often leads to treatment failure. Nonetheless, overcoming the resistance of HCC to apoptosis by inducing necroptosis of tumor cells to bypass the apoptotic pathway may be a promising treatment strategy. Sonodynamic therapy (SDT) has broad prospects in disease treatment because of its noninvasive characteristic and spatiotemporal control. The combination of SDT and shikonin in the treatment of HCC is expected to be a new tumor treatment method that can overcome apoptosis resistance., Methods: In this study, the antitumor effect was evaluated using normal liver cell line WRL68, HCC cell line HepG2 and HepG2 xenograft mouse models. Indocyanine green (ICG) was loaded on nanobubbles (NBs) to construct ICG-loaded nanobubbles (ICG-NBs). Combined sonosensitizer nanoplatforms with ultrasound (US) to achieve efficient SDT, the combination of SDT and shikonin in treating HCC can activate shikonin-induced necroptosis. As a result, tumor cells that produced apoptosis resistance were destroyed by necroptosis., Results: The results indicated a successful preparation of ICG-NBs with a uniform particle size of 273.0 ± 118.9 nm spherical structures. ICG-NB-mediated SDT, in combination with shikonin treatment, inhibited the viability, invasion, and migration of tumor cells. SDT + shikonin treatment group caused a substantial increase in necroptotic cells. The increased degree of tumor necrosis and the upregulated expression of receptor-interacting protein 3 kinase were observed in vivo studies, which indicated that the antitumor effect was accompanied by enhanced necroptosis in the SDT + shikonin treatment group., Conclusion: ICG-NB-mediated SDT combined with shikonin inhibits the growth of HCC by increasing the necroptosis of tumor cells. Therefore, this combination therapy is a promising treatment strategy against the specific cancer., Competing Interests: The authors report no conflicts of interest in this work., (© 2023 Tian et al.)

Purpose: Sonodynamic therapy (SDT) is a promising and significant measure for the treatment of tumors. However, the internal situation of hepatocellular carcinoma (HCC) is complex, separate SDT treatment is difficult to play a good therapeutic effect. Here, we used SDT combined with MG-132 to mediate apoptosis and autophagy of HCC cells to achieve the purpose of treatment of cancer., Methods: To determine the generated reactive oxygen species (ROS) and the change of mitochondrial membrane potential (ΔΨm), HepG2 cells were stained by 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) and 5,5',6,6'-Tetrachloro-1,1',3,3'-tetraethyl-imidacarbocyanine iodide (JC-1) staining to determine the IR-820@NBs-mediated SDT to achieve HCC therapy through the mitochondrial pathway. Cell counting kit 8 (CCK-8) assay and flow cytometry were used to detect cell viability and apoptosis rate of HepG2 cells. Autophagy was detected by mCherry-GFP-LC3B fluorescence labeling. Chloroquine (Cq) pretreatment was used to explore the relationship between autophagy and apoptosis. To detect the ability of HepG2 cells migration and invasion, cell scratch assay and transwell assay were used., Results: The successfully prepared IR-820@NBs could effectively overcome the shortcomings of IR-820 and induce lethal levels of ROS by ultrasound irradiation. As a dual agonist of apoptosis and autophagy, MG-132 could effectively enhance the efficacy of SDT in the process of treating HCC. After pre-treatment with Cq, the cell activity increased and the level of apoptosis decreased, which proved that apoptosis and autophagy were induced by combined therapy, autophagy, and apoptosis have the synergistic anti-tumor effect, and part of apoptosis was autophagy-dependent. After combined therapy, the activity and invasive ability of HCC cells decreased significantly., Conclusion: SDT combined with MG-132 in the process of treating liver cancer could effectively induce apoptosis and autophagy anti-tumor therapy, which is helpful to the research of new methods to treat liver cancer., Competing Interests: The authors report no conflicts of interest in this work., (© 2023 Wang et al.)

Background: Despite the clinical efficacy of immunotherapy in treating malignant tumors, its effectiveness is often hampered by the immunosuppressive nature of the tumor microenvironment (TME). In this study, we propose the design of a nanoscale ultrasound contrast agent capable of triggering macrophage polarization and immunogenic cell death (ICD) for the treatment of hepatocellular carcinoma (HCC) through sonodynamic treatment (SDT) and immunotherapy., Methods: The re-educator (designated as ICG@C3F8-R848 NBs) is composed of the Toll-like receptor agonist resiquimod (R848) and the sonosensitizer Indocyanine green (ICG), utilizing nanobubbles (NBs) as carriers. The technique known as ultrasound-targeted nanobubble destruction (UTND) employs nanosized microbubbles and low-frequency ultrasound (LFUS) to ensure accurate drug delivery and enhance safety., Results: Following intravenous delivery, ICG@C3F8-R848 NBs have the potential to selectively target and treat primary tumors using SDT in conjunction with ultrasonography. Importantly, R848 can enhance antitumor immunity by inducing the polarization of macrophages from an M2 to an M1 phenotype., Conclusion: The SDT-initiated immunotherapy utilizing ICG@C3F8-R848 NBs demonstrates significant tumor suppression effects with minimal risk of systemic toxicity. The utilization of this self-delivery re-education technique would contribute to advancing the development of nanomedicine for the treatment of hepatocellular carcinoma., Competing Interests: The authors report no conflicts of interest in this work., (© 2023 Chen et al.)

Background: The combination of sonodynamic therapy and oxygenation strategy is widely used in cancer treatment. However, due to the complexity, heterogeneity and irreversible hypoxic environment produced by hepatocellular carcinoma (HCC) tissues, oxygen-enhancing sonodynamic therapy (SDT) has failed to achieve the desired results. With the emergence of ferroptosis with reactive oxygen species (ROS) cytotoxicity, this novel cell death method has attracted widespread attention., Methods: In this study, nanobubbles (NBs) were connected with the sonosensitizer Indocyanine green (ICG) to construct a 2-in-1 nanoplatform loaded with RAS-selective lethal (RSL3, ferroptosis promoter) (RSL3@O2-ICG NBs), combined with oxygen-enhanced SDT and potent ferroptosis. In addition, nanobubbles (NBs) combined with low-frequency ultrasound (LFUS) are called ultrasound-targeted nanobubble destruction (UTND) to ensure specific drug release and improve safety., Results: MDA/GSH and other related experimental results show that RSL3@O2-ICG NBs can enhance SDT and ferroptosis. Through RNA sequencing (RNA-seq), the differential expression of LncRNA and mRNA before and after synergistic treatment was identified, and then GO and KEGG pathways were used to enrich and analyze target genes and pathways related ferroptosis sensitivity. We found that they were significantly enriched in the ferroptosis-related pathway MAPK cascade and cell proliferation. Then, we searched for the expression of differentially expressed genes in the TCGA Hepatocellular carcinoma cohort. At the same time, we evaluated the proportion of immune cell infiltration and the identification of co-expression network modules and related prognostic analysis. We found that it was significantly related to the tumor microenvironment of hepatocellular carcinoma. The prognostic risk genes "SLC37A2" and "ITGB7" may represent new hepatocellular carcinoma ferroptosis-inducing markers and have guiding significance for treating hepatocellular carcinoma., Conclusion: The therapeutic effect of the in vitro synergistic treatment has been proven to be significant, revealing the prospect of 2-in-1 nanobubbles combined with SDT and ferroptosis in treating HCC., Competing Interests: The authors report no conflicts of interest in this work., (© 2022 Chen et al.)

Long non-coding RNA (LncRNA) DUXAP9 expression was recently found to be higher in hepatocellular carcinoma (HCC) tissues and cells, and correlated with a shorter overall survival of HCC patients. However, its roles in HCC progression have never been revealed. Here, the roles of DUXAP9 in HCC cell stemness are explored as cancer stem cells (CSCs) contribute to one of the root of cancer progression. We found that DUXAP9 positively regulated HCC cell stemness, as characterized by the change of sphere-formation ability, ALDH activity and stemness marker expression. Further luciferase reporter, mRNA stability and RNA-RNA in vitro interaction assays indicated that DUXAP9 directly bound to the 3' untranslated region (UTR) of sox9, enhanced the mRNA stability of sox9 and thus increased sox9 expression. Notably, the effects induced by DUXAP9 on HCC cell stemness depended on sox9 expression. Therefore, this work identifies a novel DUXAP9/sox9 axis essential for HCC cell stemness., (© 2021 Wiley Periodicals LLC.)

Background: Ras activation is a frequent event in hepatocellular carcinoma (HCC). Combining a RAS inhibitor with traditional clinical therapeutics might be hampered by a variety of side effects, thus hindering further clinical translation. Herein, we report on integrating an IR820 nanocapsule-augmented sonodynamic therapy (SDT) with the RAS inhibitor farnesyl-thiosalicylic acid (FTS). Using cellular and tumor models, we demonstrate that combined nanocapsule-augmented SDT with FTS induces an anti-tumor effect, which not only inhibits tumor progression, and enables fluorescence imaging. To dissect the mechanism of a combined tumoricidal therapeutic strategy, we investigated the scRNA-seq transcriptional profiles of an HCC xenograft following treatment., Results: Integrative single-cell analysis identified several clusters that defined many corresponding differentially expressed genes, which provided a global view of cellular heterogeneity in HCC after combined SDT/FTS treatment. We conclude that the combination treatment suppressed HCC, and did so by inhibiting endothelial cells and a modulated immunity. Moreover, hepatic stellate secretes hepatocyte growth factor, which plays a key role in treating SDT combined FTS. By contrast, enrichment analysis estimated the functional roles of differentially expressed genes. The Gene Ontology terms "cadherin binding" and "cell adhesion molecule binding" and KEGG pathway "pathway in cancer" were significantly enriched by differentially expressed genes after combined SDT/FTS therapy., Conclusions: Thus, some undefined mechanisms were revealed by scRNA-seq analysis. This report provides a novel proof-of-concept for combinatorial HCC-targeted therapeutics that is based on a non-invasive anti-tumor therapeutic strategy and a RAS inhibitor.