Genome-wide CRISPR screenings identified SMCHD1 as a host-restricting factor for AAV transduction.

AAV-mediated gene therapy typically requires a high dose of viral transduction, risking acute immune responses and patient safety, part of which is due to limited understanding of the host-viral interactions, especially post-transduction viral genome processing. Here, through a genome-wide CRISPR screen, we identified SMCHD1 (Structural Maintenance of Chromosomes Hinge Domain 1), an epigenetic modifier, as a critical broad-spectrum restricting host factor for post-entry AAV transgene expression. SMCHD1 knock-down by RNAi and CRISPRi or knock-out by CRISPR all resulted in significantly enhanced transgene expression across multiple viral serotypes, as well as for both single-strand and self-complementary AAV genome types. Mechanistically, upon viral transduction, SMCHD1 effectively repressed AAV transcription by the formation of an LRIF1-HP1-containing protein complex and directly binding with the AAV genome to maintain a heterochromatin-like state. SMCHD1-KO or LRIF1-KD could disrupt such a complex and thus result in AAV transcriptional activation. Together, our results highlight the host factor-induced chromatin remodeling as a critical inhibitory mechanism for AAV transduction and may shed light on further improvement in AAV-based gene therapy.

Glycyrrhetinic Acid as a Hepatocyte Targeting Ligand-Functionalized Platinum(IV) Complexes for Hepatocellular Carcinoma Therapy and Overcoming Multidrug Resistance.

Promising targeted therapy options to overcome drug resistance and side effects caused by platinum(II) drugs for treatment in hepatocellular carcinoma are urgently needed. Herein, six novel multifunctional platinum(IV) complexes through linking platinum(II) agents and glycyrrhetinic acid (GA) were designed and synthesized. Among them, complex 20 showed superior antitumor activity against tested cancer cells including cisplatin resistance cells than cisplatin and simultaneously displayed good liver-targeting ability. Moreover, complex 20 can significantly cause DNA damage and mitochondrial dysfunction, promote reactive oxygen species generation, activate endoplasmic reticulum stress, and eventually induce apoptosis. Additionally, complex 20 can effectively inhibit cell migration and invasion and trigger autophagy and ferroptosis in HepG-2 cells. More importantly, complex 20 demonstrated stronger tumor inhibition ability than cisplatin or the combo of cisplatin/GA with almost no systemic toxicity in HepG-2 or A549 xenograft models. Collectively, complex 20 could be developed as a potential anti-HCC agent for cancer treatment.

Novel NF-κB Inhibitor-Conjugated Pt(IV) Prodrug to Enable Cancer Therapy through ROS/ER Stress and Mitochondrial Dysfunction and Overcome Multidrug Resistance.

Although cisplatin has been widely used for clinical purposes, its application is limited due to its obvious side effects. To mitigate the defects of cisplatin, here, six "multitarget prodrugs" were synthesized by linking cisplatin and NF-κB inhibitors. Notably, complex 9 demonstrated a 63-fold enhancement in the activity against A549/CDDP cells with lower toxicity toward normal LO2 cells compared to cisplatin. Additionally, complex 9 could effectively cause DNA damage, induce mitochondrial dysfunction, generate reactive oxygen species, and induce cell apoptosis through the mitochondrial pathway and ER stress. Remarkably, complex 9 effectively inhibited the NF-κB/MAPK signaling pathway and disrupted the PI3K/AKT signaling transduction. Importantly, complex 9 showed superior in vivo antitumor efficiency compared to cisplatin or the combination of cisplatin/4, without obvious systemic toxicity in A549 or A549/CDDP xenograft models. Our results demonstrated that the dual-acting mechanism endowed the complexes with high efficiency and low toxicity, which may represent an efficient strategy for cancer therapy.

Dual-targeting tumor cells hybrids derived from Pt(IV) species and NF-κB inhibitors enables cancer therapy through mitochondrial dysfunction and ER stress and overcomes cisplatin resistance.

To ameliorate the defects including serious side effects and drug resistance of Pt(II) drugs (e.g., cisplatin and oxaliplatin), here a novel of "dual-prodrug" by containing Pt(II) drugs and NF-κB inhibitors were synthesized and characterized. Among them, Pt(IV) complex 11 exhibited better cytotoxic activity than other Pt(IV) complexes and the corresponding Pt(II) drugs, with IC50 values ranged from 0.31 to 0.91 µM, respectively, and also displayed low toxicity toward two normal cells HL-7702 and BEAS-2B. More importantly, complex 11 significantly reversed cisplatin resistance in A549/CDDP cells, indicating that complex 11 was able to overcome multidrug resistance. Following mechanism studies demonstrated that complex 11 significantly induced DNA damage and ROS generation, arrest the cell cycle at the G2/M stage, suppressed cell migration and intrusion, and induced cell apoptosis through activated ER stress and mitochondrial apoptosis pathway in A549 cells. Moreover, complex 11 effectively suppressed the IKKß phosphorylation, IκBα phosphorylation and NF-κB p65 phosphorylation and nuclear translocation, leading to blocked the NF-κB signal pathway in A549 cells. In vivo tests showed that the inhibitory rate in the complex 11 reached 69.2 %, which was much higher than that of oxaliplatin (55.6 %), 1a (39.7 %) and the combination of oxaliplatin/1a (65.1 %), without causing loss in the body weight.

Ligustrazine-Derived Chalcones-Modified Platinum(IV) Complexes Intervene in Cisplatin Resistance in Pancreatic Cancer through Ferroptosis and Apoptosis.

Developing multitarget platinum(IV) prodrugs is an important strategy to attenuate cisplatin (CDDP) resistance in tandem with reduced toxicity. Herein, six novel ligustrazine-derived chalcones-modified platinum(IV) complexes were synthesized and evaluated for their anti-proliferative activities. Among them, 16a displayed higher cytotoxicity toward the tested cancer cell lines and lower cytotoxicity toward the human normal cells than CDDP or the combined group. Mechanistic studies revealed that 16a efficiently induced DNA damage and initiated a mitochondria-dependent apoptosis pathway. Besides, 16a significantly triggered ferroptosis by down-regulating expression levels of nuclear factor erythroid 2-related factor 2, glutathione peroxidase 4, and solute carrier family 7 member 11. Further, 16a obtained superior in vivo anti-tumor efficiency than CDDP in CDDP-resistant pancreatic cancer xenograft models but showed no significant side effects. In summary, our study suggested that 16a acts via a different anti-cancer mechanistic pathway than CDDP and may therefore encompass a novel practical strategy for cancer treatment.

Antitumor activity and mechanisms of dual EGFR/DNA-targeting strategy for the treatment of lung cancer with EGFRL858R/T790M mutation.

Dual- or multi-targeted EGFR inhibitors as single drugs can overcome EGFR inhibitor resistance and circumvent many disadvantages of combination therapy. In this work, fifteen 4-anilinoquinazoline derivatives bearing nitrogen mustard or hemi mustard moieties were designed and synthesized as dual EGFR-DNA targeting anticancer agents. Structures of target molecules were confirmed by 1H NMR, 13C NMR and HR-MS, and evaluated for their in vitro anti-proliferative activities using MTT assay. Compound 6g emerged as the most potent derivative against mutant-type H1975 cells with IC50 value of 1.45 µM, which exhibited 4-fold stronger potency than Chl/Gef (equimolar combination of chlorambucil and gefitinib). Kinase inhibition studies indicated that 6g showed excellent inhibitory effect on EGFRL858R/T790M enzyme, which was 8.6 times more effective than gefitinib. Mechanistic studies indicated that 6g induced apoptosis of H1975 cells in a dose-dependent manner and caused DNA damage. Importantly, 6g could significantly inhibit the expression of p-EGFR and its downstream p-AKT and p-ERK in H1975 cells. Molecular docking was also performed to gain insights into the ligand-binding interactions of 6g inside EGFRWT and EGFRL858R/T790M binding sites. Moreover, 6g efficiently inhibited tumor growth in the H1975 xenograft model without side effects.

Novel Indole-Chalcone Derivative-Ligated Platinum(IV) Prodrugs Attenuate Cisplatin Resistance in Lung Cancer through ROS/ER Stress and Mitochondrial Dysfunction.

Developing multifunctional platinum(IV) prodrugs via integrating bioactive pharmacophores into one entity is an attractive strategy to ameliorate the defects of platinum(II) drugs. Herein, a series of indole-chalcone derivative-ligated platinum(IV) complexes were synthesized and evaluated for their anticancer activities. Among them, optimal complex 17a exerted superior activity compared to that of cisplatin (CDDP) against the tested cells but showed lower cytotoxicity toward human normal lung cells. Detailed mechanisms demonstrated that 17a significantly enhanced intracellular accumulation, induced DNA damage, and inhibited migration in A549/CDDP cells. Furthermore, 17a efficiently disturbed the tubulin-microtubule system, initiated reactive oxygen species (ROS)-mediated endoplasmic reticulum stress, and activated a mitochondrion-dependent apoptosis signaling pathway. Besides, 17a was superior to free drugs or their combination in inhibiting cancer growth in A549/CDDP xenografts without inducing obvious side effects. The physical mixture of 16a and CDDP was almost identical to 17a but showed apparent systematic side effects. In summary, our studies may provide an efficient treatment regimen for CDDP resistance.

Novel combretastatin A-4 derivative containing aminophosphonates as dual inhibitors of tubulin and matrix metalloproteinases for lung cancer treatment.

Here, sixteen novel conjugates containing tubulin inhibitor and matrix metalloproteinase inhibitor was synthesized together with their activity evaluated. Among them, 9e exhibited the most potent activity against various human cancer cells (IC50 values was 0.19-0.42 µM) as well as multidrug-resistant tumor cells (A549/CDDP and MCF-7/DOX) and also showed significantly lower cytotoxic activity toward human normal liver cells LO2 in comparison with that of CA-4. Interestingly, 9e not only strongly inhibited tubulin polymerization, and induced cell apoptosis and cell cycle arrest in G2/M stage, but also remarkably displayed inhibition of cell migration against A549 cells in vitro, and exhibited a moderate activity toward MMP-2, MMP-3 and MMP-9, respectively. Moreover, the significant down-regulation in the levels of Bcl-2 protein and up-regulated levels of proteins, such as Bax, p53 and caspase-3, indicated that 9e can induce apoptosis via mitochondria-dependent apoptosis pathway. Additionally, 9e can also cause ER stress demonstrating as up-regulation express of proteins (CHOP, p-eIF2a, and p-PERK). Importantly, 9e displayed significant in vivo antitumor efficacy in A549 xenograft models without inducing apparent systemic toxicity. Collectively, this work indicated that compound 9e, a dual MMPs and tubulin inhibitor, is a novel and promising agent for cancer therapy.

The neurotrophic and antineuroinflammatory effects of phenylpropanoids from Zanthoxylum nitidum var. tomentosum (Rutaceae).

Three novel lignans (1, 5 and 6) and two novel quinic acids (16 and 17) along with 15 known phenylpropanoids were obtained from the ethanol extract of Zanthoxylum nitidum var. tomentosum (Rutaceae). Their structures were confirmed by comprehensive spectroscopic data (NMR and HRESIMS), and the absolute configurations of all novel compounds were elucidated based on electronic circular dichroism (ECD) spectroscopic data. The production of nitric oxide (NO) in BV-2 microglial cells induced through lipopolysaccharide (LPS) was used to evaluate in vitro anti-neuroinflammatory activity of compounds 1-20. Compound 2, 3, 7 and 16 showed excellent inhibition of LPS-induced NO production. The structure-activity relationships of the isolates were investigated. In addition, the mechanism of action of 2 was elucidated by RT-PCR and Western blotting analysis, which indicated that it reduced neuroinflammatory mainly through NLRP3/caspase1 signaling pathways in LPS-induced BV2 microglial cells.

Dual-target platinum(IV) complexes exhibit antiproliferative activity through DNA damage and induce ER-stress-mediated apoptosis in A549 cells.

Platinum(II)-based chemotherapeutics are commonly used to treat various types of solid tumors, such as lung cancers. However, these compounds can cause serious side effects, including nephrotoxicity and ototoxicity, which affect the quality of life of patients. In our work, four novel dual target platinum(IV) complexes were designed and synthesized. In vitro results indicated that the title platinum(IV) complexes exhibited effective antitumor activities against the tested cancer cells and had lower toxicity and resistance factors than oxaliplatin and cisplatin. Further mechanistic experiments demonstrated that complex 11 accumulated in mitochondria and induced an elevation in ROS and an ER stress response via mitochondrial dysfunction. Notably, complex 11 significantly modulated the expression levels of proapoptosis proteins including cleaved-Caspase-3, Bax, and p53, and decreased the level of the prosurvival protein Bcl-2. Together, these results suggested that complex 11 might be a potential lead compound for future cancer therapy due to its potency and selectivity.

Platinum(IV) complexes conjugated with chalcone analogs as dual targeting anticancer agents: In vitro and in vivo studies.

For the sake to develop novel platinum(IV) complexes to reverse cisplatin (CDDP) resistence, four multifunctional platinum(IV) prodrugs via conjugating chalcones with the related platinum(IV) complexes derived from cisplatin were designed and evaluated for anti-tumor actyivities in vitro and in vivo. Among them, complex 9 exhibited excellent anticancer activities in vitro with IC50 values at the submicromolar level against the tested human cancer cells, whereas showed low cytotoxicity towards human normal liver cells HL-7702. Further mechanistic studies indicated that complex 9 induced G2/M phase arrest and apoptosis in A549 cells, which was associated with a collapse of the mitochondrial membrane potential (MMP), alterations in the expression of some apoptosis-related proteins, and enhanced level of the intracellular reactive oxygen species (ROS). More importantly, complex 9 significantly suppressed the tumor growth in the A549 xenograft model without obvious hints of toxicity.

Bifunctional Naphthoquinone Aromatic Amide-Oxime Derivatives Exert Combined Immunotherapeutic and Antitumor Effects through Simultaneous Targeting of Indoleamine-2,3-dioxygenase and Signal Transducer and Activator of Transcription 3.

Indoleamine-2,3-dioxygenase 1 (IDO1) and signal transducer and activator of transcription 3 (STAT3) are important targets in the tumor microenvironment for cancer therapy. In the present study, a set of naphthoquinone aromatic amide-oxime derivatives were designed, which stimulated the immune response via IDO1 inhibition and simultaneously displayed powerful antitumor activity against three selected cancer cell lines through suppressing STAT3 signaling. The representative compound 8u bound effectively to IDO1, with greater inhibitory activity relative to the commercial IDO1 inhibitor 4-amino-N-(3-chloro-4-fluorophenyl)-N'-hydroxy-1,2,5-oxadiazole-3-carboximidamide (IDO5L) in addition to the efficient suppression of nuclear translocation of STAT3. Consistently, in vivo assays demonstrated a higher antiproliferative activity of compound 8u in both wild-type B16-F10 isograft tumors and an athymic HepG2 xenograft model relative to 1-methyl-l-tryptophan (1-MT) and doxorubicin (DOX). This bifunctional compound with dual immunotherapeutic and anticancer efficacy may represent a new generation of highly efficacious drug candidates for cancer therapy.

Synthesis, mechanisms of action, and toxicity of novel aminophosphonates derivatives conjugated irinotecan in vitro and in vivo as potent antitumor agents.

Twenty novel aminophosphonates derivatives (5a-5j and 6a-6j) conjugated irinotecan were synthesized through esterification reaction, and evaluated their anticancer activities using MTT assay. In vitro evaluation revealed that they displayed similar or superior cytotoxicity compared to the positive drug irinotecan against A549, MCF-7, SK-OV-3, MG-63, U2OS and multidrug-resistant (MDR) SK-OV-3/CDDP cancer cell lines. Among them, 9b displayed the most potent activity, with IC50 values of 0.92-3.23 µM against five human cancer cells, which exhibited a 5.4-19.1-fold increase in activity compared to the reference drug irinotecan, respectively. Moreover, cellular mechanism studies suggested that 9b arrested cell cycle at S stage and induced cell apoptosis along with the decrease of mitochondrial membrane potential (MMP). Interestingly, 9b significantly inhibited tumor growth in SK-OV-3 xenograft models in vivo without apparent toxicity, which was better than the positive drug irinotecan. Taken together, 9b possessed potent antitumor activity and may be a promising candidate for the potential treatment of human ovarian cancer cells.

Synthesis and biological evaluation of novel millepachine derivative containing aminophosphonate ester species as novel anti-tubulin agents.

A new series of millepachine derivative containing aminophosphonate ester moieties were designed and synthesized, and evaluated for their anticancer activities using MTT assay. Among all the compounds, compound 9m exhibited the most potent cytotoxic activity against all tested human cancer cell lines including multidrug resistant phenotype, which inhibited cancer cell growth with IC50 values ranging from 0.85 to 3.09 µM, respectively. In addition, its low cytotoxicity toward human normal liver cells HL-7702 and sensitivity toward to doxorubicin or cisplatin-resistant cells indicated the possibility for cancer therapy. Furthermore, 9m significantly induced cell apoptosis and cell cycle arrest in G2/M phase and dramatically disrupted the microtubule organization. Moreover, a decrease in MMP, an increase in reactive oxygen species (ROS) generation and Bax/Bcl-2 ratio, accompanied by activated caspase-3 and -9, were observed in HepG-2 cells after incubation with 9m, indicating that the mitochondrial pathway was involved in the 9m-mediated apoptosis.

Dual-targeting antitumor conjugates derived from platinum(IV) prodrugs and microtubule inhibitor CA-4 significantly exhibited potent ability to overcome cisplatin resistance.

Here we report that three platinum(IV) prodrugs containing a tubulin inhibitor CA-4, as dual-targeting platinum(IV) prodrug, were synthesized and evaluated for antitumor activity using MTT assay. Among them, complex 9 exhibited the most potent antitumor activity against the tested cancer lines including cisplatin resistance cancer cells, and simultaneously displayed lower toxicity compared to cisplatin, respectively. Moreover, complex 9, in which was conjugated to an inhibitor of tubulin at one axial position of platinum(IV) complex, could effectively enter the cancer cells, and significantly induce cell apoptosis and arrest the cell cycle in A549 cells at G2/M stage, and dramatically disrupt the microtubule organization. In addition, mechanism studies suggested that complex 9 significantly induced reactive oxygen species (ROS) generation and decreased mitochondrial trans-membrane potential (MMP) in A549 cells, and effectively induced activation of caspases triggering apoptotic signaling through mitochondrial dependent apoptosis pathways.

Promoting antitumor efficacy by suppressing hypoxia via nano self-assembly of two irinotecan-based dual drug conjugates having a HIF-1α inhibitor.

Hypoxia inducible factor (HIF-1α), as a major transcription factor in response to hypoxia, revealed that it could be a promising tumor-specific target for anticancer therapy. In view of clinical application, the formation of a hypoxic microenvironment in tumors can decrease the curative effect of cytotoxic chemotherapeutic drugs. To promote the antitumor efficacy of chemical drugs by suppressing hypoxia, we designed and conjugated a hydrophobic HIF-1α inhibitor (YC-1) to a hydrophilic anticancer drug, irinotecan (Ir), into one molecular entity via dicarboxylate and PEG3 linkages. Benefiting from its amphiphilicity, the resulting conjugate could act as molecular building blocks to self-assemble into nanoparticles (Ir-YC-1 and Ir-PEG3-YC-1 NPs) in water, which improved the solubility of drugs. As expected, the Ir-YC-1 NPs significantly down-regulated the expression of HIF-1α and VEGF proteins, and exhibited 5.7-fold increased antitumor activity compared to Ir when administered to A549 cells under the hypoxic condition. This novel, simple, and effective strategy for overcoming tumor hypoxia and enhancing the antitumor effect of chemical drugs has great potential in cancer therapy.

Glycyrrhetinic acid derivatives containing aminophosphonate ester species as multidrug resistance reversers that block the NF-κB pathway and cell proliferation.

Novel NF-κB inhibitors based on Glycyrrhetinic acid (GA) derivatives containing aminophosphonate ester moieties were rationally designed and synthesized as well as evaluated their antitumor activities using MTT assay. Many target compounds showed potent antitumor activities against the tested human cancer cell lines including cisplatin-resistant cells, and exhibited significant inhibitory activity to the NF-κB with IC50 values at micromolar concentrations in A549 cells, respectively. Among them, compound 12e possessed excellent antitumor activities against the tested human cancer cell lines and showed low cytotoxicity toward to human normal liver cells. Moreover, 12e caused obvious loss of MMP and significantly induced ROS production, and displayed inhibition of cell migration against A549 cells in vitro. Importantly, 12e arrested the cell cycle at the S phases and ultimately induced cell apoptosis in A549 cells through blockage of NF-κB signaling pathway. Our research provided an efficient strategy for targeting NF-κB antitumor drug development.

Pt(IV) prodrugs containing microtubule inhibitors displayed potent antitumor activity and ability to overcome cisplatin resistance.

It is well-known that cisplatin exhibited a broad spectrum of anticancer activities against many solid tumors, but its severe toxicity and drug resistance have largely limited wider clinical applications. Various strategies have been tried to discover new Pt (II) drugs with at least equal activity as well as low toxicity compared to cisplatin, but the inherent problem remains unsolved. Here we report that Pt (IV) complexes comprising a CA-4 analogue, as dual-targeting Pt (IV) prodrug, were synthesized and evaluated for anti-proliferative activity using MTT assay. Among them, complex 19 displayed most potent activity against the tested cancer cell lines, and simultaneously exhibited better cell selectivity between cancer cells and normal cells than that of cisplatin. Mechanism studies revealed that complex 19 effectively induced cell cycle arrest at the G2/M phase and dramatically disrupted the microtubule organization. Moreover, complex 19 significantly induced cell apoptosis and decreased MMP. Importantly, complex 19 significantly inhibited tumor growth in SK-OV-3 xenograft model in vivo without apparent toxicity.

Discovery of antitumor ursolic acid long-chain diamine derivatives as potent inhibitors of NF-κB.

A series of inhibitors of NF-κB based on ursolic acid (UA) derivatives containing long-chain diamine moieties were designed and synthesized as well as evaluated the antitumor effects. These compounds exhibited significant inhibitory activity to the NF-κB with IC50 values at micromolar concentrations in A549 lung cancer cell line. Among them, compound 8c exerted potent activity against the test tumor cell lines including multidrug resistant human cancer lines, with the IC50 values ranged from 5.22 to 8.95 µM. Moreover, compound 8c successfully suppressed the migration of A549 cells. Related mechanism study indicated compound 8c caused cell cycle arrest at G1 phase and triggered apoptosis in A549 cells through blockage of NF-κB signalling pathway. Molecular docking study revealed that key interactions between 8c and the active site of NF-κB in which the bulky and strongly electrophilic group of long-chain diamine moieties were important for improving activity.

16-O-caffeoyl-16-hydroxylhexadecanoic acid, a medicinal plant-derived phenylpropanoid, induces apoptosis in human hepatocarcinoma cells through ROS-dependent endoplasmic reticulum stress.

BACKGROUND: Hepatocellular carcinoma (HCC) is the leading cause of cancer death, and novel chemotherapeutic drugs for treating HCC are urgently needed. 16-O-caffeoyl-16-hydroxylhexadecanoic acid (CHHA) is a new phenylpropanoid isolated by our group from Euphorbia nematocypha which is commonly used to treat solid tumors. However, the underlying mechanisms responsible for the CHHA-induced apoptosis in cancer cells, particularly in HCC, remain unknown. PURPOSE: In the present work, we evaluated the growth inhibitory effect of CHHA on HCC cells and explored the underlying molecular mechanisms. METHODS/STUDY DESIGNS: The anti-proliferative activity of CHHA was evaluated by MTT assay. Cell cycle, apoptosis, reactive oxygen species and mitochondrial membrane potential were determined by flow cytometry. ER localization was performed by ER-tracker red staining. The effect of CHHA on the expression of mRNA in HCC cells was detected by RT-PCR. The potential mechanisms for proteins level in ER pathway and apoptosis were analyzed by Western blot. RESULTS: Our results showed that CHHA exerted strong anti-proliferative activity against both HepG2 and Bel-7402 cells in a concentration- and time-dependent manner. Mechanistic studies demonstrated that CHHA induced apoptosis through mitochondrial apoptotic pathway, and arrested the cell cycle at G1 phase. CHHA was also found to induce endoplasmic reticulum (ER) stress, accompanied by ROS production, increase of intracellular calcium and up-regulation of GRP78, CHOP, caspase-12 and p-PERK. Inhibition of endoplasmic reticulum stress by salubrinal pretreatment could suppress both apoptosis and ER stress, indicating that ER stress induction contributes to apoptosis and is required for the latter. Besides, the ROS scavenger N-acetyl cysteine (NAC) significantly attenuated apoptosis induced by CHHA and reversed CHHA-stimulated the expression of ER markers. CONCLUSION: In conclusion, CHHA inhibited HCC cell growth and induced apoptosis through mitochondria-mediated pathway and ROS-mediated endoplasmic reticulum stress. This provides molecular bases for developing CHHA into a drug candidate for the treatment of HCC.