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Abstract Background Cancer is characterized by dysregulated cellular metabolism. Thus, understanding the mechanisms underlying these metabolic alterations is important for developing targeted therapies. In this study, we investigated the pro-tumoral effect of PDZ and LIM domain 2 (PDLIM2) downregulation in lung cancer growth and its association with the accumulation of mitochondrial ROS, oncometabolites and the activation of hypoxia-inducible factor-1 (HIF-1) α in the process. Methods Databases and human cancer tissue samples were analyzed to investigate the roles of PDLIM2 and HIF-1α in cancer growth. DNA microarray and gene ontology enrichment analyses were performed to determine the cellular functions of PDLIM2. Seahorse assay, flow cytometric analysis, and confocal microscopic analysis were employed to study mitochondrial functions. Oncometabolites were analyzed using liquid chromatography–mass spectrometry (LC–MS). A Lewis lung carcinoma (LLC) mouse model was established to assess the in vivo function of PDLIM2 and HIF-1α. Results The expression of PDLIM2 was downregulated in lung cancer, and this downregulation correlated with poor prognosis in patients. PDLIM2 highly regulated genes associated with mitochondrial functions. Mechanistically, PDLIM2 downregulation resulted in NF-κB activation, impaired expression of tricarboxylic acid (TCA) cycle genes particularly the succinate dehydrogenase (SDH) genes, and mitochondrial dysfunction. This disturbance contributed to the accumulation of succinate and other oncometabolites, as well as the buildup of mitochondrial reactive oxygen species (mtROS), leading to the activation of hypoxia-inducible factor 1α (HIF-1α). Furthermore, the expression of HIF-1α was increased in all stages of lung cancer. The expression of PDLIM2 and HIF-1α was reversely correlated in lung cancer patients. In the animal study, the orally administered HIF-1α inhibitor, PX-478, significantly reduces PDLIM2 knockdown-promoted tumor growth. Conclusion These findings shed light on the complex action of PDLIM2 on mitochondria and HIF-1α activities in lung cancer, emphasizing the role of HIF-1α in the tumor-promoting effect of PDLIM2 downregulation. Additionally, they provide new insights into a strategy for precise targeted treatment by suggesting that HIF-1α inhibitors may serve as therapy for lung cancer patients with PDLIM2 downregulation.

Colorectal cancer (CRC) is one of the primary contributors to cancer-related fatalities, with up to 80% of advanced CRC cases exhibiting mutations in the p53 gene. Unfortunately, the development of new compounds targeting mutant p53 is quite limited. The anticancer effects of Dehydroepiandrosterone (DHEA) on various cancers have been reported. However, the suppressive effect of DHEA on CRC cells harboring wild-type or mutant p53 gene remains controversial. This study emphasized revealing the suppressive mechanism and the effect of DHEA on CRC cell tumorigenesis in the presence of wild-type or mutant p53 gene. We demonstrate that DHEA causes CRC cell death and cell cycle arrest in a dose and time-dependent manner. Notably, DHEA exhibits similar inhibitory effects on CRC cells regardless of the p53 gene status. Further study reveals that DHEA induces endoplasmic reticulum (ER) stress and triggers PERK/eIF2/ATF4/CHOP UPR signaling pathway to activate autophagy followed by apoptosis, which was confirmed by suppression of 4-phenylbutyric acid (an ER stress inhibitor) or knockdown either ATF4 or CHOP. DHEA-induced apoptosis was attenuated by silencing ATG5 gene in either p53+/+ or p53−/− CRC cells, indicating autophagy regulation of apoptosis. Furthermore, DHEA treatment accompanied by bafilomycin A1 (a blocker of autophagosome degradation) leads to the accumulation of ATF4, CHOP, DR5, and p21 levels in CRC cells, implying that the degradative autophagy machinery regulates these four molecules. Consistently, DHEA demonstrates its inhibitory effect by suppressing CRC tumor formation in vivo. Altogether, we provide compelling evidence that DHEA is a potential therapeutic candidate for CRC patient treatment regardless of the p53 status through ER stress-PERK-autophagy-apoptosis axis.

Abstract Background Acute lung injury (ALI) is a life-threatening respiratory condition characterized by severe inflammation and lung tissue damage, frequently causing rapid respiratory failure and long-term complications. The microRNA let-7a-5p is involved in the progression of lung injury, inflammation, and fibrosis by regulating immune cell activation and cytokine production. This study aims to use an innovative cellular electroporation platform to generate extracellular vesicles (EVs) carring let-7a-5p (EV-let-7a-5p) derived from transfected Wharton’s jelly-mesenchymal stem cells (WJ-MSCs) as a potential gene therapy for ALI. Methods A cellular nanoporation (CNP) method was used to induce the production and release of EV-let-7a-5p from WJ-MSCs transfected with the relevant plasmid DNA. EV-let-7a-5p in the conditioned medium were isolated using a tangential flow filtration (TFF) system. EV characterization followed the minimal consensus guidelines outlined by the International Society for Extracellular Vesicles. We conducted a thorough set of therapeutic assessments, including the antifibrotic effects using a transforming growth factor beta (TGF-β)-induced cell model, the modulation effects on macrophage polarization, and the influence of EV-let-7a-5p in a rat model of hyperoxia-induced ALI. Results The CNP platform significantly increased EV secretion from transfected WJ-MSCs, and the encapsulated let-7a-5p in engineered EVs was markedly higher than that in untreated WJ-MSCs. These EV-let-7a-5p did not influence cell proliferation and effectively mitigated the TGF-β-induced fibrotic phenotype by downregulating SMAD2/3 phosphorylation in LL29 cells. Furthermore, EV-let-7a-5p regulated M2-like macrophage activation in an inflammatory microenvironment and significantly induced interleukin (IL)-10 secretion, demonstrating their modulatory effect on inflammation. Administering EVs from untreated WJ-MSCs slightly improved lung function and increased let-7a-5p expression in plasma in the hyperoxia-induced ALI rat model. In comparison, EV-let-7a-5p significantly reduced macrophage infiltration and collagen deposition while increasing IL-10 expression, causing a substantial improvement in lung function. Conclusion This study reveals that the use of the CNP platform to stimulate and transfect WJ-MSCs could generate an abundance of let-7a-5p-enriched EVs, which underscores the therapeutic potential in countering inflammatory responses, fibrotic activation, and hyperoxia-induced lung injury. These results provide potential avenues for developing innovative therapeutic approaches for more effective interventions in ALI.

Objectives: Determining a diagnosis for non-Tuberculous mycobacterium (NTM)-lung disease (LD) remains difficult. The value of circulating cell-free DNA (cfDNA) secreted from microbes has been established in the detection of pathogens in septic patients. However, it is unknown whether NTM-derived cfDNA is detectable in plasma from patients with NTM-LD and whether this is associated with the disease status of NTM-LD, especially in patients with Mycobacterium avium complex (MAC)-LD. Materials and Methods: In this pilot study, from 2018 to 2019, we enrolled adult patients with MAC-LD at Taipei Veterans General Hospital in Taiwan for the detection of circulating cfDNA. We performed cfDNA extraction from plasma, next-generation sequencing (NGS) for nonhuman cfDNA, and sequence matching to a microbial database and then assessed the association between pathogen cfDNA and MAC-LD. Results: Two (40%) plasma samples from MAC-LD patients had detectable MAC-specific cfDNA, namely one instance of DNA polymerase III alpha subunit and one instance of ATP-binding cassette transporters permease. The plasma samples from the three other MAC-LD cases and the one tuberculosis control were negative for either NTM-derived cfDNA or tuberculosis-related cfDNA. In addition to MAC-specific cfDNA, Ralstonia solanacearum, Staphylococcus aureus, and Pasteurella multocida were the most observed bacteria in our patients. The two patients with MAC-cfDNA positivity yielded higher radiographic scores (P = 0.076) and presented a higher number of nonhuman reads than those without MAC-cfDNA positivity (P = 0.083). Conclusion: Using NGS method, we demonstrated MAC-cfDNA was detectable in patients with MAC-LD. Further large-scale research is warranted to assess the clinical value of detecting MAC-specific cfDNA in MAC-LD patients.

Abstract Detecting pancreatic duct adenocarcinoma (PDAC) in its early stages and predicting late‐stage patient prognosis undergoing chemotherapy is challenging. This work shows that the activation of specific oncogenes leads to elevated expression of mRNAs and their corresponding proteins in extracellular vesicles (EVs) circulating in blood. Utilizing an immune lipoplex nanoparticle (ILN) biochip assay, these findings demonstrate that glypican 1 (GPC1) mRNA expression in the exosomes‐rich (Exo) EV subpopulation and GPC1 membrane protein (mProtein) expression in the microvesicles‐rich (MV) EV subpopulation, particularly the tumor associated microvesicles (tMV), served as a viable biomarker for PDAC. A combined analysis effectively discriminated early‐stage PDAC patients from benign pancreatic diseases and healthy donors in sizable clinical from multiple hospitals. Furthermore, among late‐stage PDAC patients undergoing chemotherapy, lower GPC1 tMV‐mProtein and Exo‐mRNA expression before treatment correlated significantly with prolonged overall survival. These findings underscore the potential of vesicular GPC1 expression for early PDAC screenings and chemotherapy prognosis.

Abstract Pancreatic ductal adenocarcinoma (PDAC) tumours carry multiple gene mutations and respond poorly to treatments. There is currently an unmet need for drug carriers that can deliver multiple gene cargoes to target high solid tumour burden like PDAC. Here, we report a dual targeted extracellular vesicle (dtEV) carrying high loads of therapeutic RNA that effectively suppresses large PDAC tumours in mice. The EV surface contains a CD64 protein that has a tissue targeting peptide and a humanized monoclonal antibody. Cells sequentially transfected with plasmid DNAs encoding for the RNA and protein of interest by Transwell®-based asymmetric cell electroporation release abundant targeted EVs with high RNA loading. Together with a low dose chemotherapy drug, Gemcitabine, dtEVs suppress large orthotopic PANC-1 and patient derived xenograft tumours and metastasis in mice and extended animal survival. Our work presents a clinically accessible and scalable way to produce abundant EVs for delivering multiple gene cargoes to large solid tumours.

Abstract Background Colorectal cancer (CRC) is a highly prevalent cancer type with limited targeted therapies available and 5-year survival rate, particularly for late-stage patients. There have been numerous attempts to repurpose drugs to tackle this problem. It has been reported that autophagy inducers could augment the effect of certain chemotherapeutic agents by enhancing immunogenic cell death (ICD). Methods In this study, we employed bioinformatics tools to identify thioridazine (THD), an antipsychotic drug, and found that it could induce autophagy and ICD in CRC. Then in vitro and in vivo experiments were performed to further elucidate the molecular mechanism of THD in CRC. Results THD was found to induce endoplasmic reticulum (ER) stress in CRC cells by activating the eIF2α/ATF4/CHOP axis and facilitating the accumulation of secretory autophagosomes, leading to ICD. In addition, THD showed a remarkable ICD-activating effect when combined with oxaliplatin (OXA) to prevent tumor progression in the mouse model. Conclusions Together, our findings suggest that the repurposed function of THD in inhibiting CRC involves the upregulation of autophagosomes and ER stress signals, promoting the release of ICD markers, and providing a potential candidate to enhance the clinical outcome for CRC treatment. Video Abstract

Abstract Background Despite advances in treatment, patients with refractory colorectal cancer (CRC) still have poor long-term survival, so there is a need for more effective therapeutic options. Methods To evaluate the HDAC8 inhibition efficacy as a CRC treatment, we examined the effects of various HDAC8 inhibitors (HDAC8i), including BMX (NBM-T-L-BMX-OS01) in combination with temozolomide (TMZ) or other standard CRC drugs on p53 mutated HT29 cells, as well as wild-type p53 HCT116 and RKO cells. Results We showed that HDAC8i with TMZ cotreatment resulted in HT29 arrest in the S and G2/M phase, whereas HCT116 and RKO arrest in the G0/G1 phase was accompanied by high sub-G1. Subsequently, this combination approach upregulated p53-mediated MGMT inhibition, leading to apoptosis. Furthermore, we observed the cotreatment also enabled triggering of cell senescence and decreased expression of stem cell biomarkers. Mechanistically, we found down-expression levels of β-catenin, cyclin D1 and c-Myc via GSK3β/β-catenin signaling. Intriguingly, autophagy also contributes to cell death under the opposite status of β-catenin/p62 axis, suggesting that there exists a negative feedback regulation between Wnt/β-catenin and autophagy. Consistently, the Gene Set Enrichment Analysis (GSEA) indicated both apoptotic and autophagy biomarkers in HT29 and RKO were upregulated after treating with BMX. Conclusions BMX may act as a HDAC8 eraser and in combination with reframed-TMZ generates a remarkable synergic effect, providing a novel therapeutic target for various CRCs. Video Abstract

Identifying essential targets in the genome-scale metabolic networks of cancer cells is a time-consuming process. The present study proposed a fuzzy hierarchical optimization framework for identifying essential genes, metabolites and reactions. On the basis of four objectives, the present study developed a framework for identifying essential targets that lead to cancer cell death and evaluating metabolic flux perturbations in normal cells that have been caused by cancer treatment. Through fuzzy set theory, a multiobjective optimization problem was converted into a trilevel maximizing decision-making (MDM) problem. We applied nested hybrid differential evolution to solve the trilevel MDM problem to identify essential targets in genome-scale metabolic models for five consensus molecular subtypes (CMSs) of colorectal cancer. We used various media to identify essential targets for each CMS and discovered that most targets affected all five CMSs and that some genes were CMS-specific. We obtained experimental data on the lethality of cancer cell lines from the DepMap database to validate the identified essential genes. The results reveal that most of the identified essential genes were compatible with the colorectal cancer cell lines obtained from DepMap and that these genes, with the exception of EBP, LSS, and SLC7A6, could generate a high level of cell death when knocked out. The identified essential genes were mostly involved in cholesterol biosynthesis, nucleotide metabolisms, and the glycerophospholipid biosynthetic pathway. The genes involved in the cholesterol biosynthetic pathway were also revealed to be determinable, if a cholesterol uptake reaction was not induced when the cells were in the culture medium. However, the genes involved in the cholesterol biosynthetic pathway became non-essential if such a reaction was induced. Furthermore, the essential gene CRLS1 was revealed as a medium-independent target for all CMSs.

The immune checkpoint inhibitor/tyrosine kinase inhibitor (ICI/TKI) combination treatment is currently the first-line treatment for metastatic renal cell carcinoma (mRCC). However, its efficacy beyond the third-line setting is expected to be relatively poor, and high-grade toxicities can develop by prior exposure to multiple drugs, resulting in a relatively poor performance in patients. Determining the best treatment regimen and sequence remains difficult and requires further investigation in patients with mRCC. In this study, two cases of mRCC, who failed several lines of TKI and nivolumab but exhibited a good anticancer effect after rechallenging with axitinib, are described. Both patients had a faster time to best response and better progression-free survival (PFS) than during previous treatments. Moreover, the axitinib dose could be reduced to 2.5 mg daily when used in combination with nivolumab while continuing to exert an impressive anticancer effect. To determine the cytotoxic effect, we performed a lymphocyte activation test and found that the level of granzyme B released by cytotoxic T lymphocytes and natural killer cells was higher when axitinib was combined with nivolumab. To evaluate this result, a bioinformatics approach was used to analyze the PRISM database. In conclusion, based on the results of a lymphocyte activation test and PD-1 expression, our findings indicate that sequential therapy with axitinib rechallenge after nivolumab resistance is reasonable for the treatment of mRCC.

Coronavirus disease 2019 (COVID-19) remains a threat with the emergence of new variants, especially Delta and Omicron, without specific effective therapeutic drugs. The infection causes dysregulation of the immune system with a cytokine storm that eventually leads to fatal acute respiratory distress syndrome (ARDS) and further irreversible pulmonary fibrosis. Therefore, the promising way to inhibit infection is to disrupt the binding and fusion between the viral spike and the host ACE2 receptor. A transcriptome-based drug screening platform has been developed for COVID-19 to explore the possibility and potential of the long-established drugs or herbal medicines to reverse the unique genetic signature of COVID-19. In silico analysis showed that Virofree, an herbal medicine, reversed the genetic signature of COVID-19 and ARDS. Biochemical validations showed that Virofree could disrupt the binding of wild-type and Delta-variant spike proteins to ACE2 and its syncytial formation via cell-based pseudo-typed viral assays, as well as suppress binding between several variant recombinant spikes to ACE2, especially Delta and Omicron. Additionally, Virofree elevated miR-148b-5p levels, inhibited the main protease of SARS-CoV-2 (Mpro), and reduced LPS-induced TNF-α release. Virofree also prevented cellular iron accumulation leading to ferroptosis which occurs in SARS-CoV-2 patients. Furthermore, Virofree was able to reduce pulmonary fibrosis-related protein expression levels in vitro. In conclusion, Virofree was repurposed as a potential herbal medicine to combat COVID-19. This study highlights the inhibitory effect of Virofree on the entry of Delta and Omicron variants of SARS-CoV-2, which have not had any effective treatments during the emergence of the new variants spreading.

PurposeCurrent treatment options for head and neck squamous cell carcinoma (HNSCC) are limited, especially for cases with cancer stem cell-induced chemoresistance and recurrence. The WNT signaling pathway contributes to maintenance of stemness via translocation of β-catenin into the nucleus, and represents a promising druggable target in HNSCC. Dehydroepiandrosterone (DHEA), a steroid hormone, has potential as an anticancer drug. However, the potential anticancer mechanisms of DHEA including inhibition of stemness, and its therapeutic applications in HNSCC remain unclear.MethodsFirstly, SRB assay and sphere formation assay were used to examine cellular viability and cancer stem cell-like phenotype, respectively. The expressions of stemness related factors were measured by RT-qPCR and western blotting. The luciferase reporter assay was applied to evaluate transcriptional potential of stemness related pathways. The alternations of WNT signaling pathway were measured by nuclear translocation of β-catenin, RT-qPCR and western blotting. Furthermore, to investigate the effect of drugs in vivo, both HNSCC orthotopic and subcutaneous xenograft mouse models were applied.ResultsWe found that DHEA reduced HNSCC cell viability, suppressed sphere formation, and inhibited the expression of cancer-stemness markers, such as BMI-1 and Nestin. Moreover, DHEA repressed the transcriptional activity of stemness-related pathways. In the WNT pathway, DHEA reduced the nuclear translocation of the active form of β-catenin and reduced the protein expression of the downstream targets, CCND1 and CD44. Furthermore, when combined with the chemotherapeutic drug, irinotecan (IRN), DHEA enhanced the sensitivity of HNSCC cells to IRN as revealed by reduced cell viability, sphere formation, expression of stemness markers, and activation of the WNT pathway. Additionally, this combination reduced in vivo tumor growth in both orthotopic and subcutaneous xenograft mouse models.ConclusionThese findings indicate that DHEA has anti-stemness potential in HNSCC and serves as a promising anticancer agent. The combination of DHEA and IRN may provide a potential therapeutic strategy for patients with advanced HNSCC.

BackgroundFactor V (FV) deficiency is a rare disease, with a low incidence rate in Asia. Therefore, the F5 mutation in the Taiwanese population is poorly understood.MethodsA Chinese family with FV deficiency was included, and the patient and his family members underwent mutation analysis. Then, patients from Keelung City (Taiwan) were screened for F5 polymorphism; the Chang Gung Human Database was used to determine single-nucleotide variants in the non-FV-deficient patient population.ResultsEight mutation sites on the F5 gene locus, including exon 16 homozygote Met1736Val and seven heterozygous mutations, including Asp68His, were found. Moreover, Met1736Val was found to be the dominant mutation in people living in the Taiwan community, and this result was compared with the records of the Chang Gung Human Database. The above-mentioned polymorphisms may result in a variable incidence of FV deficiency in Keelung City, thereby facilitating carrier diagnosis and prenatal diagnosis in most FV-deficient families.ConclusionThe homozygote Met1736Val and the co-inheritance of the Asp68His F5 gene are unique and worthy of screening in FV-deficient patients.

The outbreak of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses a serious threat to global public health. In an effort to develop novel anti-coronavirus therapeutics and achieve prophylactics, we used gene set enrichment analysis (GSEA) for drug screening and identified that Astragalus polysaccharide (PG2), a mixture of polysaccharides purified from Astragalus membranaceus, could effectively reverse COVID-19 signature genes. Further biological assays revealed that PG2 could prevent the fusion of BHK21-expressing wild-type (WT) viral spike (S) protein and Calu-3-expressing ACE2. Additionally, it specifically prevents the binding of recombinant viral S of WT, alpha, and beta strains to ACE2 receptor in our non-cell-based system. In addition, PG2 enhances let-7a, miR-146a, and miR-148b expression levels in the lung epithelial cells. These findings speculate that PG2 has the potential to reduce viral replication in lung and cytokine storm via these PG2-induced miRNAs. Furthermore, macrophage activation is one of the primary issues leading to the complicated condition of COVID-19 patients, and our results revealed that PG2 could regulate the activation of macrophages by promoting the polarization of THP-1-derived macrophages into an anti-inflammatory phenotype. In this study, PG2 stimulated M2 macrophage activation and increased the expression levels of anti-inflammatory cytokines IL-10 and IL-1RN. Additionally, PG2 was recently used to treat patients with severe COVID-19 symptoms by reducing the neutrophil-to-lymphocyte ratio (NLR). Therefore, our data suggest that PG2, a repurposed drug, possesses the potential to prevent WT SARS-CoV-2 S-mediated syncytia formation with the host cells; it also inhibits the binding of S proteins of WT, alpha, and beta strains to the recombinant ACE2 and halts severe COVID-19 development by regulating the polarization of macrophages to M2 cells.

COVID-19 is threatening human health worldwide but no effective treatment currently exists for this disease. Current therapeutic strategies focus on the inhibition of viral replication or using anti-inflammatory/immunomodulatory compounds to improve host immunity, but not both. Traditional Chinese medicine (TCM) compounds could be promising candidates due to their safety and minimal toxicity. In this study, we have developed a novel in silico bioinformatics workflow that integrates multiple databases to predict the use of honeysuckle (Lonicera japonica) and Huangqi (Astragalus membranaceus) as potential anti-SARS-CoV-2 agents. Using extracts from honeysuckle and Huangqi, these two herbs upregulated a group of microRNAs including let-7a, miR-148b, and miR-146a, which are critical to reduce the pathogenesis of SARS-CoV-2. Moreover, these herbs suppressed pro-inflammatory cytokines including IL-6 or TNF-α, which were both identified in the cytokine storm of acute respiratory distress syndrome, a major cause of COVID-19 death. Furthermore, both herbs partially inhibited the fusion of SARS-CoV-2 spike protein-transfected BHK-21 cells with the human lung cancer cell line Calu-3 that was expressing ACE2 receptors. These herbs inhibited SARS-CoV-2 Mpro activity, thereby alleviating viral entry as well as replication. In conclusion, our findings demonstrate that honeysuckle and Huangqi have the potential to be used as an inhibitor of SARS-CoV-2 virus entry that warrants further in vivo analysis and functional assessment of miRNAs to confirm their clinical importance. This fast-screening platform can also be applied to other drug discovery studies for other infectious diseases.

Abstract Background Ovarian cancer is the leading cause of cancer-related death among women. Complete cytoreductive surgery followed by platinum-taxene chemotherapy has been the gold standard for a long time. Various compounds have been assessed in an attempt to combine them with conventional chemotherapy to improve survival rates or even overcome chemoresistance. Many studies have shown that an antidiabetic drug, metformin, has cytotoxic activity in different cancer models. However, the synergism of metformin as a neoadjuvant formula plus chemotherapy in clinical trials and basic studies remains unclear for ovarian cancer. Methods We applied two clinical databases to survey metformin use and ovarian cancer survival rate. The Cancer Genome Atlas dataset, an L1000 microarray with Gene Set Enrichment Analysis (GSEA) analysis, Western blot analysis and an animal model were used to study the activity of the AKT/mTOR pathway in response to the synergistic effects of neoadjuvant metformin combined with chemotherapy. Results We found that ovarian cancer patients treated with metformin had significantly longer overall survival than patients treated without metformin. The protein profile induced by low- concentration metformin in ovarian cancer predominantly involved the AKT/mTOR pathway. In combination with chemotherapy, the neoadjuvant metformin protocol showed beneficial synergistic effects in vitro and in vivo. Conclusions This study shows that neoadjuvant metformin at clinically relevant dosages is efficacious in treating ovarian cancer, and the results can be used to guide clinical trials.

ALDH2 gene is coded for the aldehyde dehydrogenase (ALDH), which is an enzyme involved in alcohol metabolism. Compared to normal aldehyde dehydrogenases, a homozygous point mutation on exon 12 from G to A significantly reduces its efficiency. In this study, we have reported the generation of IBMS-iPSC-021-04, IBMS-iPSC-022-01, and IBMS-iPSC-023-03 as induced pluripotent stem cell (iPSC) lines carrying the homozygous form of ALDH2 with the rs671 genetic polymorphism (E487K mutation). These cell lines were characterized in terms of pluripotency and differentiation potential. They serve as useful platforms to study alcohol metabolism and other chronic diseases associated with alcohol consumption.

Atrial fibrillation is the most common heart disease in the world, with around 35 million patients in 2020. Here we reported the generation of IBMS-iPSC-015-06, IBMS-iPSC-016-06, and IBMS-iPSC-017-02 as human induced pluripotent stem cell (iPSC) lines from patients’ peripheral blood mononuclear cells (PBMCs) with atrial fibrillation. The cell lines expressed properties of pluripotent stem cells, including pluripotent markers and the ability to differentiate into three germ layers. These cell lines served as suitable models for studying alternative therapies of atrial fibrillation.

P-glycoprotein (also known as multidrug resistance protein 1 (MDR1) or ATP-binding cassette sub-family B member 1 (ABCB1) plays a crucial role in determining response against medications, including cancer therapeutics. It is now well established that p-glycoprotein acts as an ATP dependent pump that pumps out small molecules from cells. Ample evidence exist that show p-glycoprotein expression levels correlate with drug efficacy, which suggests the rationale for developing p-glycoprotein inhibitors for treatment against cancer. Preclinical and clinical studies have investigated this possibility, but mostly were limited by substantial toxicities. Repurposing FDA-approved drugs that have p-glycoprotein inhibition activities is therefore a potential alternative approach. In this review, we searched the Drugbank Database (https://www.drugbank.ca/drugs) and identified 98 FDA-approved small molecules that possess p-glycoprotein inhibition properties. Focusing on the small molecules approved with indications against non-cancer diseases, we query the scientific literature for studies that specifically investigate these therapeutics as cancer treatment. In light of this analysis, potential development opportunities will then be thoroughly investigated for future efforts in repositioning of non-cancer p-glycoprotein inhibitors in single use or in combination therapy for clinical oncology treatment.

Infrapatellar fat pad–derived mesenchymal stem cells (IPFP-MSCs) are a type of adipose-derived stem cell (ADSC). They potentially contribute to cartilage regeneration and modulation of the immune microenvironment in patients with osteoarthritis (OA). The ability of IPFP-MSCs to increase chondrogenic capacity has been reported to be greater, less age dependent, and less affected by inflammatory changes than that of other MSCs. Transcription-regulatory factors strictly regulate the cartilage differentiation of MSCs. However, few studies have explored the effect of transcriptional factors on IPFP-MSC-based neocartilage formation, cartilage engineering, and tissue functionality during and after chondrogenesis. Instead of intact MSCs, MSC-derived extracellular vesicles could be used for the treatment of OA. Furthermore, exosomes are increasingly being considered the principal therapeutic agent in MSC secretions that is responsible for the regenerative and immunomodulatory functions of MSCs in cartilage repair. The present study provides an overview of advancements in enhancement strategies for IPFP-MSC chondrogenic differentiation, including the effects of transcriptional factors, the modulation of released exosomes, delivery mechanisms for MSCs, and ethical and regulatory points concerning the development of MSC products. This review will contribute to the understanding of the IPFP-MSC chondrogenic differentiation process and enable the improvement of IPFP-MSC-based cartilage tissue engineering.

Vaccines are powerful tools for controlling microbial infections and preventing epidemic diseases. Efficient inactive, subunit, or viral-like particle vaccines usually rely on a safe and potent adjuvant to boost the immune response to the antigen. After a slow start, over the last decade there has been increased developments on adjuvants for human vaccines. The development of adjuvants has paralleled our increased understanding of the molecular mechanisms for the pattern recognition receptor (PRR)-mediated activation of immune responses. Toll-like receptors (TLRs) are a group of PRRs that recognize microbial pathogens to initiate a host’s response to infection. Activation of TLRs triggers potent and immediate innate immune responses, which leads to subsequent adaptive immune responses. Therefore, these TLRs are ideal targets for the development of effective adjuvants. To date, TLR agonists such as monophosphoryl lipid A (MPL) and CpG-1018 have been formulated in licensed vaccines for their adjuvant activity, and other TLR agonists are being developed for this purpose. The COVID-19 pandemic has also accelerated clinical research of vaccines containing TLR agonist-based adjuvants. In this paper, we reviewed the agonists for TLR activation and the molecular mechanisms associated with the adjuvants’ effects on TLR activation, emphasizing recent advances in the development of TLR agonist-based vaccine adjuvants for infectious diseases.

Immunotherapy using checkpoint blockade has revolutionized cancer treatment, improving patient survival and quality of life. Nevertheless, the clinical outcomes of such immunotherapy are highly heterogeneous between patients. Depending on the cancer type, the patient response rates to this immunotherapy are limited to 20–30%. Based on the mechanism underlying the antitumor immune response, new therapeutic strategies have been designed with the aim of increasing the effectiveness and specificity of the antitumor immune response elicited by checkpoint blockade agents. The activation of toll-like receptor 9 (TLR9) by its synthetic agonists induces the antitumor response within the innate immunity arm, generating adjuvant effects and priming the adaptive immune response elicited by checkpoint blockade during the effector phase of tumor-cell killing. This review first describes the underlying mechanisms of action and current status of monotherapy using TLR9 agonists and immune checkpoint inhibitors for cancer immunotherapy. The rationale for combining these two agents is discussed, and evidence indicating the current status of such combination therapy as a novel cancer treatment strategy is presented.

Cancer cells are known to exhibit unusual metabolic activity, and yet few metabolic cancer driver genes are known. Genetic alterations and epigenetic modifications of cancer cells result in the abnormal regulation of cellular metabolic pathways that are different when compared with normal cells. Such a metabolic reprogramming can be simulated using constraint-based modelling approaches towards predicting oncogenes. We introduced the tri-level optimization problem to use the metabolic reprogramming towards inferring oncogenes. The algorithm incorporated Recon 2.2 network with the Human Protein Atlas to reconstruct genome-scale metabolic network models of the tissue-specific cells at normal and cancer states, respectively. Such reconstructed models were applied to build the templates of the metabolic reprogramming between normal and cancer cell metabolism. The inference optimization problem was formulated to use the templates as a measure towards predicting oncogenes. The nested hybrid differential evolution algorithm was applied to solve the problem to overcome solving difficulty for transferring the inner optimization problem into the single one. Head and neck squamous cells were applied as a case study to evaluate the algorithm. We detected 13 of the top-ranked one-hit dysregulations and 17 of the top-ranked two-hit oncogenes with high similarity ratios to the templates. According to the literature survey, most inferred oncogenes are consistent with the observation in various tissues. Furthermore, the inferred oncogenes were highly connected with the TP53/AKT/IGF/MTOR signalling pathway through PTEN, which is one of the most frequently detected tumour suppressor genes in human cancer.

Abstract Overexpression of ALDH is associated with cancer stem-like features and poor cancer prognosis. High ALDH activity has been observed in cancer stem-like cells. There are a total of 19 human ALDH isoforms, all of which are associated with reducing oxidative stress and protecting cells from damage. However, it is unknown whether all ALDHs are associated with poor cancer prognosis and which ones play a significant role in cancer progression. In this study, we used RNA sequencing data from The Cancer Genome Atlas (TCGA) to evaluate the differential expression of 19 ALDH isoforms in 5 common human cancers. The 19 ALDH genes were analyzed with an integrating meta-analysis of cancer prognosis. Genotyping and next-generation RNA sequencing for 30 pairwise samples of head and neck squamous cell carcinoma were performed and compared with the TCGA cohort. The analysis showed that each ALDH isoform had a specific differential expression pattern, most of which were related to prognosis in human cancer. A lower expression of ALDH2 in the tumor was observed, which was independent from the ALDH2 rs671 SNP variant and the expression of other mitochondria-associated protein coding genes. This study provides new insight into the association between ALDH expression and cancer prognosis.

Chronic myeloid leukemia (CML) is a hematopoietic malignancy characterized by the presence of the BCR-ABL oncogene. Therapeutic regimens with tyrosine kinase inhibitors (TKIs) specifically targeting BCR-ABL have greatly improved overall survival of CML. However, drug intolerance and related toxicity remain. Combined therapy is effective in reducing drug magnitude while increasing therapeutic efficacy and, thus, lowers undesired adverse side effects. The p38 MAPK activity is critically linked to the pathogenesis of a number of diseases including hematopoietic diseases; however, the role of each isozyme in CML and TKI-mediated effects is still elusive. In this study, we used specific gene knockdown to clearly demonstrate that the deficiency of p38α greatly enhanced the therapeutic efficacy in growth suppression and cytotoxicity of TKIs, first-generation imatinib, and second generation dasatinib by approximately 2.5–3.0-fold in BCR-ABL-positive CML-derived leukemia K562 and KMB5 cells. Knockdown of p38β, which displays the most sequence similarity to p38α, exerted distinct and opposite effects on the TKI-mediated therapeutic efficacy. These results show the importance of isotype-specific intervention in enhancing the therapeutic efficacy of TKI. A highly specific p38α inhibitor, TAK715, also significantly enhanced the imatinib- and dasatinib-mediated therapeutic efficacy, supporting the feasibility of p38α deficiency in future clinic application. Taken together, our results demonstrated that p38α is a promising target for combined therapy with BCR-ABL-targeting tyrosine kinase inhibitors for future application to increase therapeutic efficacy.

Declines in physiological functions are the predominant risk factors for age-related diseases, such as cancers and neurodegenerative diseases. Therefore, delaying the aging process is believed to be beneficial in preventing the onset of age-related diseases. Previous studies have demonstrated that Graptopetalum paraguayense (GP) extract inhibits liver cancer cell growth and reduces the pathological phenotypes of Alzheimer’s disease (AD) in patient IPS-derived neurons. Here, we show that GP extract suppresses β-amyloid pathology in SH-SYS5Y-APP695 cells and APP/PS1 mice. Moreover, AMP-activated protein kinase (AMPK) activity is enhanced by GP extract in U87 cells and APP/PS1 mice. Intriguingly, GP extract enhances autophagy in SH-SYS5Y-APP695 cells, U87 cells, and the nematode Caenorhabditis elegans, suggesting a conserved molecular mechanism by which GP extract might regulate autophagy. In agreement with its role as an autophagy activator, GP extract markedly diminishes mobility decline in polyglutamine Q35 mutants and aged wild-type N2 animals in C. elegans. Furthermore, GP extract significantly extends lifespan in C. elegans.

The efficient discovery of anticancer targets with minimal side effects is a major challenge in drug discovery and development. Early prediction of side effects is key for reducing development costs, increasing drug efficacy, and increasing drug safety. This study developed a fuzzy optimization framework for Identifying AntiCancer Targets (IACT) using constraint-based models. Four objectives were established to evaluate the mortality of treated cancer cells and to minimize side effects causing toxicity-induced tumorigenesis on normal cells and smaller metabolic perturbations. Fuzzy set theory was applied to evaluate potential side effects and investigate the magnitude of metabolic deviations in perturbed cells compared with their normal counterparts. The framework was applied to identify not only gene regulator targets but also metabolite- and reaction-centric targets. A nested hybrid differential evolution algorithm with a hierarchical fitness function was applied to solve multilevel IACT problems. The results show that the combination of a carbon metabolism target and any one-target gene that participates in the sphingolipid, glycerophospholipid, nucleotide, cholesterol biosynthesis, or pentose phosphate pathways is more effective for treatment than one-target inhibition is. A clinical antimetabolite drug 5-fluorouracil (5-FU) has been used to inhibit synthesis of deoxythymidine-5′-triphosphate for treatment of colorectal cancer. The computational results reveal that a two-target combination of 5-FU and a folate supplement can improve cell viability, reduce metabolic deviation, and reduce side effects of normal cells.

The multi-domain non-structural protein 3 (NSP3) is an oncogenic molecule that has been concomitantly implicated in the progression of coronavirus infection. However, its oncological role in lung cancer and whether it plays a role in modulating the tumor immune microenvironment is not properly understood. In the present in silico study, we demonstrated that NSP3 (SH2D3C) is associated with advanced stage and poor prognoses of lung cancer cohorts. Genetic alterations of NSP3 (SH2D3C) co-occurred inversely with Epidermal Growth Factor Receptor (EGFR) alterations and elicited its pathological role via modulation of various components of the immune and inflammatory pathways in lung cancer. Our correlation analysis suggested that NSP3 (SH2D3C) promotes tumor immune evasion via dysfunctional T-cell phenotypes and T-cell exclusion mechanisms in lung cancer patients. NSP3 (SH2D3C) demonstrated a high predictive value and association with therapy resistance in lung cancer, hence serving as an attractive target for therapy exploration. We evaluated the in silico drug-likeness and NSP3 (SH2D3C) target efficacy of six organosulfur small molecules from Allium sativum using a molecular docking study. We found that the six organosulfur compounds demonstrated selective cytotoxic potential against cancer cell lines and good predictions for ADMET properties, drug-likeness, and safety profile. E-ajoene, alliin, diallyl sulfide, 2-vinyl-4H-1,3-dithiin, allicin, and S-allyl-cysteine docked well into the NSP3 (SH2D3C)-binding cavity with binding affinities ranging from –4.3~–6.70 Ă and random forest (RF) scores ranging from 4.31~5.26 pKd. However, S-allyl-cysteine interaction with NSP3 (SH2D3C) is unfavorable and hence less susceptible to NSP3 ligandability. In conclusion, our study revealed that NSP3 is an important onco-immunological biomarker encompassing the tumor microenvironment, disease staging and prognosis in lung cancer and could serve as an attractive target for cancer therapy. The organosulfur compounds from A. sativum have molecular properties to efficiently interact with the binding site of NSP3 and are currently under vigorous preclinical study in our laboratory.

Cholangiocarcinoma is the most common primary malignant tumor of the bile duct. The current standard first-line treatment for advanced or metastatic cholangiocarcinoma is gemcitabine and cisplatin. However, few effective treatment choices exist for refractory cholangiocarcinoma, and additional therapeutic drugs are urgently required. Our previous work demonstrated that the ALDH isoform 1A3 plays a vital role in the malignant behavior of cholangiocarcinoma and may serve as a new therapeutic target. In this study, we found a positive correlation between ALDH1A3 protein expression levels and the cell migration abilities of three cholangiocarcinoma cell lines, which was verified using ALDH1A3-overexpressing and ALDH1A3-knockdown clones. We also used ALDH1A3-high and ALDH1A3-low populations of cholangiocarcinoma cell lines from the library of integrated network-based cellular signatures (LINCS) program and assessed the effects of ruxolitinib, a commercially available JAK2 inhibitor. Ruxolitinib had a higher cytotoxic effect when combined with gemcitabine. Furthermore, the nuclear translocation STAT1 and STAT3 heterodimers were markedly diminished by ruxolitinib treatment, possibly resulting in decreased ALDH1A3 activation. Notably, ruxolitinib alone or combined with gemcitabine led to significantly reduced tumor size and weight. Collectively, our studies suggest that ruxolitinib might suppress the ALDH1A3 activation through the JAK2/STAT1/3 pathway in cholangiocarcinoma, and trials should be undertaken to evaluate its efficacy in clinical therapy.

Nonarteritic anterior ischemic optic neuropathy (NAION) is the most common cause of acute vision loss in older people, and there is no effective therapy. The effect of the systemic or local application of steroids for NAION patients remains controversial. Oroxylin A (OA) (5,7-dihydroxy-6-methoxyflavone) is a bioactive flavonoid extracted from Scutellariae baicalensis Georgi. with various beneficial effects, including anti-inflammatory and neuroprotective effects. A previous study showed that OA promotes retinal ganglion cell (RGC) survival after optic nerve (ON) crush injury. The purpose of this research was to further explore the potential actions of OA in ischemic injury in an experimental anterior ischemic optic neuropathy (rAION) rat model induced by photothrombosis. Our results show that OA efficiently attenuated ischemic injury in rats by reducing optic disc edema, the apoptotic death of retinal ganglion cells, and the infiltration of inflammatory cells. Moreover, OA significantly ameliorated the pathologic changes of demyelination, modulated microglial polarization, and preserved visual function after rAION induction. OA activated nuclear factor E2 related factor (Nrf2) signaling and its downstream antioxidant enzymes NAD(P)H:quinone oxidoreductase (NQO-1) and heme oxygenase 1 (HO-1) in the retina. We demonstrated that OA activates Nrf2 signaling, protecting retinal ganglion cells from ischemic injury, in the rAION model and could potentially be used as a therapeutic approach in ischemic optic neuropathy.

Although histone deacetylase 8 (HDAC8) plays a role in glioblastoma multiforme (GBM), whether its inhibition facilitates the treatment of temozolomide (TMZ)-resistant GBM (GBM-R) remains unclear. By assessing the gene expression profiles from short hairpin RNA of HDAC8 in the new version of Connectivity Map (CLUE) and cells treated by NBM-BMX (BMX)-, an HDAC8 inhibitor, data analysis reveals that the Wnt signaling pathway and apoptosis might be the underlying mechanisms in BMX-elicited treatment. This study evaluated the efficacy of cotreatment with BMX and TMZ in GBM-R cells. We observed that cotreatment with BMX and TMZ could overcome resistance in GBM-R cells and inhibit cell viability, markedly inhibit cell proliferation, and then induce cell cycle arrest and apoptosis. In addition, the expression level of β-catenin was reversed by proteasome inhibitor via the β-catenin/ GSK3β signaling pathway to reduce the expression level of c-Myc and cyclin D1 in GBM-R cells. BMX and TMZ cotreatment also upregulated WT-p53 mediated MGMT inhibition, thereby triggering the activation of caspase-3 and eventually leading to apoptosis in GBM-R cells. Moreover, BMX and TMZ attenuated the expression of CD133, CD44, and SOX2 in GBM-R cells. In conclusion, BMX overcomes TMZ resistance by enhancing TMZ-mediated cytotoxic effect by downregulating the β-catenin/c-Myc/SOX2 signaling pathway and upregulating WT-p53 mediated MGMT inhibition. These findings indicate a promising drug combination for precision personal treating of TMZ-resistant WT-p53 GBM cells.

Therapeutic resistance in recurrent glioblastoma multiforme (GBM) after concurrent chemoradiotherapy (CCRT) is a challenging issue. Although standard fractionated radiation is essential to treat GBM, it has led to local recurrence along with therapy-resistant cells in the ionizing radiation (IR) field. Lines of evidence showed cancer stem cells (CSCs) play a vital role in therapy resistance in many cancer types, including GBM. However, the molecular mechanism is poorly understood. Here, we proposed that autophagy could be involved in GSC induction for radioresistance. In a clinical setting, patients who received radiation/chemotherapy had higher LC3II expression and showed poor overall survival compared with those with low LC3 II. In a cell model, U87MG and GBM8401 expressed high level of stemness markers CD133, CD44, Nestin, and autophagy marker P62/LC3II after receiving standard fractionated IR. Furthermore, Wnt/β-catenin proved to be a potential pathway and related to P62 by using proteasome inhibitor (MG132). Moreover, pharmacological inhibition of autophagy with BAF and CQ inhibit GSC cell growth by impairing autophagy flux as demonstrated by decrease Nestin, CD133, and SOX-2 levels. In conclusion, we demonstrated that fractionated IR could induce GSCs with the stemness phenotype by P62-mediated autophagy through the Wnt/β-catenin for radioresistance. This study offers a new therapeutic strategy for targeting GBM in the future.

Justicidin A (JA), a natural lignan, was isolated from the whole plant of Justicia procumbens, one of the most popular traditional Chinese medicines in China and Taiwan. Gene expression signatures of JA from human colorectal cancer HT-29 and hepatocellular carcinoma Hep 3B cells were used to query connectivity map. A NF-κB suppressor (15-delta prostaglandin J2) was predicted to display similar molecular action of JA. In JA-treated HT-29 cells, suppression of nuclear NF-κB expression and decrease of NF-κB DNA-binding activity were indeed observed. The classical pathway was involved in JA-induced inhibition of NF-κB, characterized by decreases in phosphorylation of AKT, IκB kinase (IKK)-β/IKK-α, and IκB-α. Furthermore, JA caused significant translocation of apoptosis-inducing factor and endonuclease G, caspase-independent apoptotic signaling molecules, from the mitochondrial to the nuclei. Our data suggest anti-inflammatory and cytotoxic mechanisms of JA, and using gene expression signatures to identify novel molecular actions of phytochemicals is an effective approach.

Mitochondrial fission and fusion cycles are integrated with cell cycle progression. Here we first re-visited how mitochondrial ETC inhibition disturbed mitosis progression, resulting in multipolar spindles formation in HeLa cells. Inhibitors of ETC complex I (rotenone, ROT) and complex III (antimycin A, AA) decreased the phosphorylation of Plk1 T210 and Aurora A T288 in the mitotic phase (M-phase), especially ROT, affecting the dynamic phosphorylation status of fission protein dynamin-related protein 1 (Drp1) and the Ser637/Ser616 ratio. We then tested whether specific Drp1 inhibitors, Mdivi-1 or Dynasore, affected the dynamic phosphorylation status of Drp1. Similar to the effects of ROT and AA, our results showed that Mdivi-1 but not Dynasore influenced the dynamic phosphorylation status of Ser637 and Ser616 in Drp1, which converged with mitotic kinases (Cdk1, Plk1, Aurora A) and centrosome-associated proteins to significantly accelerate mitotic defects. Moreover, our data also indicated that evoking mito-Drp1-Ser637 by protein kinase A (PKA) rather than Drp1-Ser616 by Cdk1/Cyclin B resulted in mitochondrial fission via the PINK1/Parkin pathway to promote more efficient mitophagy and simultaneously caused multipolar spindles. Collectively, this study is the first to uncover that mito-Drp1-Ser637 by PKA, but not Drp1-Ser616, drives mitophagy to exert multipolar spindles formation during M-phase.

Honeysuckle (Lonicera japonica Thunb) is a traditional Chinese medicine (TCM) with an antipathogenic activity. MicroRNAs (miRNAs) are small non-coding RNA molecules that are ubiquitously expressed in cells. Endogenous miRNA may function as an innate response to block pathogen invasion. The miRNA expression profiles of both mice and humans after the ingestion of honeysuckle were obtained. Fifteen overexpressed miRNAs overlapped and were predicted to be capable of targeting three viruses: dengue virus (DENV), enterovirus 71 (EV71) and SARS-CoV-2. Among them, let-7a was examined to be capable of targeting the EV71 RNA genome by reporter assay and Western blotting. Moreover, honeysuckle-induced let-7a suppression of EV71 RNA and protein expression as well as viral replication were investigated both in vitro and in vivo. We demonstrated that let-7a targeted EV71 at the predicted sequences using luciferase reporter plasmids as well as two infectious replicons (pMP4-y-5 and pTOPO-4643). The suppression of EV71 replication and viral load was demonstrated in two cell lines by luciferase activity, RT-PCR, real-time PCR, Western blotting and plaque assay. Furthermore, EV71-infected suckling mice fed honeysuckle extract or inoculated with let-7a showed decreased clinical scores and a prolonged survival time accompanied with decreased viral RNA, protein expression and virus titer. The ingestion of honeysuckle attenuates EV71 replication and related pathogenesis partially through the upregulation of let-7a expression both in vitro and in vivo. Our previous report and the current findings imply that both honeysuckle and upregulated let-7a can execute a suppressive function against the replication of DENV and EV71. Taken together, this evidence indicates that honeysuckle can induce the expression of let-7a and that this miRNA as well as 11 other miRNAs have great potential to prevent and suppress EV71 replication.

The tautomeric pair of garcinielliptone FC (GFC) is a pure and structure-defined active ingredient, isolated from Garcinia subelliptica Merr., a plant with abundant sources of polyphenols. Here, we demonstrate that a higher concentration of GFC had no effect on normal human umbilical vein endothelial cells. On chemoresistant human colorectal cancer HT-29 cells, a time- and dosage-related cytotoxicity was observed. GFC also inhibited the nuclear expression and DNA binding activity of nuclear factor κB in a time- and dosage-related manner using Western blot and electrophoretic mobility shift assay. Furthermore, GFC induced apoptosis, which was characterized by an increase of cells at the sub-G1 phase, induction of DNA fragmentation, and formation of apoptotic bodies using flow cytometry, electrophoresis, and fluorescence microscopy, respectively. Western blot analysis revealed that both caspase-dependent intrinsic and extrinsic pathways as well as caspase-independent pathway were involved. Our data suggest the anticancer mechanisms of GFC on human colorectal cancer.

Justicidin A (JA), a novel arylnaphthalide lignan, was previously found to reduce the growth of human hepatocellular carcinoma cells (HCC), Hep 3B cells, in NOD-SCID mice via induction of apoptosis. Here, using L1000 microarray profiling obtained from JA-treated HCC cells, the z-score signatures of JA exhibited positive connectivity to known autophagy inducers among 3122 compounds from the Library of Integrated Network-based Cellular Signatures database. JA-induced autophagy in HCC Hep 3B cells was confirmed by formation of acidic vesicular organelles and an increase in the expression of LC3-II and LAMP2a. JA-induced autophagic flux was demonstrated by accumulation of LC3-II and LAMP2a, as well as a decrease in colocalization of LC3 and LAMP2a puncta in the presence of autophagy inhibitor bafilomycin A1. Administration of bafilomycin A1 also demonstrated that JA-induced autophagy suppressed apoptosis. Activation of Ras/MEK/ERK pathway was observed. Following administration of apoptosis inhibitor zVAD, it was found that JA-induced apoptosis did not affect JA-induced autophagy. It is noteworthy that addition of JA promoted chemosensitivity of sorafenib, the only FDA-approved targeted therapy for advanced HCC. The results demonstrate the effectiveness of this novel strategy for rapid discovery of molecular actions of small molecules and suggest the applications of JA in HCC treatment.