Tissue-resident memory and circulating T cells are early responders to pre-surgical cancer immunotherapy.

Neoadjuvant immune checkpoint blockade has shown promising clinical activity. Here, we characterized early kinetics in tumor-infiltrating and circulating immune cells in oral cancer patients treated with neoadjuvant anti-PD-1 or anti-PD-1/CTLA-4 in a clinical trial (NCT02919683). Tumor-infiltrating CD8 T cells that clonally expanded during immunotherapy expressed elevated tissue-resident memory and cytotoxicity programs, which were already active prior to therapy, supporting the capacity for rapid response. Systematic target discovery revealed that treatment-expanded tumor T cell clones in responding patients recognized several self-antigens, including the cancer-specific antigen MAGEA1. Treatment also induced a systemic immune response characterized by expansion of activated T cells enriched for tumor-infiltrating T cell clonotypes, including both pre-existing and emergent clonotypes undetectable prior to therapy. The frequency of activated blood CD8 T cells, notably pre-treatment PD-1-positive KLRG1-negative T cells, was strongly associated with intra-tumoral pathological response. These results demonstrate how neoadjuvant checkpoint blockade induces local and systemic tumor immunity.

Genetic modifiers of the BRD4-NUT dependency of NUT midline carcinoma uncovers a synergism between BETis and CDK4/6is.

Bromodomain and extraterminal (BET) domain inhibitors (BETis) show efficacy on NUT midline carcinoma (NMC). However, not all NMC patients respond, and responders eventually develop resistance and relapse. Using CRISPR and ORF expression screens, we systematically examined the ability of cancer drivers to mediate resistance of NMC to BETis and uncovered six general classes/pathways mediating resistance. Among these, we showed that RRAS2 attenuated the effect of JQ1 in part by sustaining ERK pathway function during BRD4 inhibition. Furthermore, overexpression of Kruppel-like factor 4 (KLF4), mediated BETi resistance in NMC cells through restoration of the E2F and MYC gene expression program. Finally, we found that expression of cyclin D1 or an oncogenic cyclin D3 mutant or RB1 loss protected NMC cells from BETi-induced cell cycle arrest. Consistent with these findings, cyclin-dependent kinase 4/6 (CDK4/6) inhibitors showed synergistic effects with BETis on NMC in vitro as well as in vivo, thereby establishing a potential two-drug therapy for NMC.

A genetic interaction analysis identifies cancer drivers that modify EGFR dependency.

A large number of cancer drivers have been identified through tumor sequencing efforts, but how they interact and the degree to which they can substitute for each other have not been systematically explored. To comprehensively investigate how cancer drivers genetically interact, we searched for modifiers of epidermal growth factor receptor (EGFR) dependency by performing CRISPR, shRNA, and expression screens in a non-small cell lung cancer (NSCLC) model. We elucidated a broad spectrum of tumor suppressor genes (TSGs) and oncogenes (OGs) that can genetically modify proliferation and survival of cancer cells when EGFR signaling is altered. These include genes already known to mediate EGFR inhibitor resistance as well as many TSGs not previously connected to EGFR and whose biological functions in tumorigenesis are not well understood. We show that mutation of PBRM1, a subunit of the SWI/SNF complex, attenuates the effects of EGFR inhibition in part by sustaining AKT signaling. We also show that mutation of Capicua (CIC), a transcriptional repressor, suppresses the effects of EGFR inhibition by partially restoring the EGFR-promoted gene expression program, including the sustained expression of Ets transcription factors such as ETV1 Together, our data provide strong support for the hypothesis that many cancer drivers can substitute for each other in certain contexts and broaden our understanding of EGFR regulation.

Evaluation of Dose-Response Relationship of Permeation Enhancer Isopropyl Myristate Release on Drug Release: Release Enhancement Efficiency and Molecular Mechanism.

The objective of this study is to investigate the dose-response relationship between various concentrations of permeation enhancers (PEs) and their ability to enhance drug release from a polymer matrix, utilizing an innovative parameter known as release enhancement efficiency (K). Additionally, the molecular mechanism underlying dynamic enhancement was also examined. Isopropyl myristate (IPM) was used as model enhancer and zolmitriptan (ZOL) was used as model drug to investigate dose-effect relationship in pressure sensitive adhesives (PSA). The release behavior of the PEs was determined by LC-MS/MS and verified by confocal laser scanning microscopy (CLSM). The enhancing effect of the PE on ZOL release was evaluated through in vitro release experiments and further validated by pharmacokinetics study. And the molecular mechanism was characterized with thermal analysis (DSC), Fourier transform infrared spectroscopy (FT-IR) and molecular dynamics simulation. K was 0.156, 0.286 and 0.279 at 3%, 6% and 9% IPM concentrations, indicating that the enhancement efficiency reached the maximum when the 6% IPM was applied. According to the mechanism research results, the fluidity of PSA increased linearly with the increase of IPM concentrations, but the interaction between IPM and ZOL reached its strongest point at 6%. In summary, the increase of K value (from 0 to 6% IPM content) was caused by the synergy of increased mobility of PSA and interaction (dipole-dipole and hydrogen-bond) among three components, and when the above two actions were in antagonistic, K no longer increased (6-9% IPM content).

The effect of skin diffusion kinetics of isopropyl ester permeation enhancers on drug permeation: Role of lateral spread and penetration characteristics.

The purpose of present work was to study the effects of permeation enhancers' two kinetic behaviors of simultaneous lateral diffusion and vertical penetration in the skin on its enhancing effect. The skin diffusion kinetics of isopropyl ester permeation enhancers were characterized by the innovative concentric tape peeling study and Raman imaging, which were quantitatively assessed through innovative parameters, namely, lateral-to-vertical penetration amount (CL-V) and lateral-to-vertical penetration distance (DL-V). The enhancement effect of permeation enhancers on drug flurbiprofen (FLU) was assessed by in vitro skin permeation tests, which were confirmed by transdermal water loss and skin resistance study. The relationship between kinetic parameters of permeation enhancers and permeation parameters of FLU was carried out by correlation analysis. The molecular mechanisms of effect of skin diffusion kinetics of permeation enhancers on drug permeation were characterized by molecular docking, modulated-temperature differential scanning calorimetry (MTDSC), Raman spectra, solid-state NMR and molecular dynamic simulation. The results indicated skin diffusion kinetics of short-chain (C8-C12) isopropyl ester permeation enhancers were governed by vertical penetration, while long-chain (C14-C18) ones were characterized by lateral spread. Quadratic correlation between CL-V and enhancement ratio of permeation-retention ratio of FLU (ERQ/R) (R2 = 0.95), DL-V and enhancement ratio of permeation area (ERA) of FLU (R2 = 0.98) indicating that varied skin diffusion kinetics of permeation enhancers directly influenced the barrier function of stratum corneum (SC) and further enhancing drug permeation. In terms of molecular mechanism, long-chain isopropyl ester enhancers had good miscibility with SC, leading to their high CL-V and DL-V, and causing strong interaction strength with SC and resulting in weaker skin barrier function for drug permeation. In summary, in comparison to short-chain isopropyl ester enhancers that relied on penetration, long-chain ones that depended on lateral spread exhibited greater enhancement efficacy, which guided the application of enhancers in transdermal formulations.

Quanzhen Yiqi decoction attenuates inflammation in mice with smoking-induced COPD by activating the Nrf2/HO-1 pathway and inhibiting the NLRP3 inflammasome.

OBJECTIVE: Quanzhen Yiqi decoction (QZYQ) is a traditional Chinese medicine for treating chronic obstructive pulmonary disease. METHODS: Mice were exposed to cigarette smoke (CS) 6 days/week (40 cigarettes/day) for 24 weeks and then intragastrically administered QZYQ (4.72, 9.45, or 18.89 g/kg) or dexamethasone (DEX, 0.6 mg/kg) for 6 weeks. We examined the lung function and collected bronchoalveolar lavage fluid for inflammatory cell and cytokine quantification. The pathological lung changes, ROS and oxidative biomarkers were measured. We used immunohistochemistry and western blotting to evaluate the levels of Nrf2/HO-1, NLRP3/ASC/Caspase1/IL-1ß/IL-18. RESULTS: The CS group showed significant increases in the forced vital capacity, lung resistance, and chord compliance and a lower FEV50/FVC compared with the control, and QZYQ improved these changes. In addition, QZYQ effectively reduced emphysema, immune cell infiltration, and airway remodeling. QZYQ stimulated HO-1 expression and reduced oxidative stress through the Nrf2 pathway. QZYQ inhibited the production of NLRP3/ASC/Caspase-1 to inhibit IL-1ß and IL-18. CONCLUSION: Our study suggested that QZYQ can improve the function and histology of the lungs and reduce inflammatory cell recruitment. QZYQ inhibits ROS production and NLRP3 inflammasome activation by upregulating Nrf2 to reduce lung injury. The anti-inflammatory effects of QZYQ are similar to those of DEX.

Schisandrin A regulates the Nrf2 signaling pathway and inhibits NLRP3 inflammasome activation to interfere with pyroptosis in a mouse model of COPD.

Chronic obstructive pulmonary disease (COPD) is a serious chronic lung disease. Schisandrin A (SchA) is one of the most important active ingredients in Schisandra chinensis and has been used to treat various lung diseases in several countries. Here, we studied the pharmacological effect of SchA on airway inflammation induced by cigarette smoke (CS) and explored the therapeutic mechanism of SchA in COPD model mice. Our results showed that SchA treatment significantly improved the lung function of CS-induced COPD model mice and reduced the recruitment of leukocytes and hypersecretion of interleukin-6 (IL-6), interleukin-1ß (IL-1ß) and tumor necrosis factor α (TNF-α) in bronchoalveolar lavage fluid (BALF). H&E staining showed that SchA treatment could effectively reduce emphysema, immune cell infiltration and airway wall destruction. In addition, we found that SchA treatment can stimulate the expression of heme oxygenase-1 (HO-1) through the nuclear factor-erythroid 2-related factor (Nrf2) pathway, significantly reduce oxidative stress, increase catalase (CAT) and superoxide dismutase (SOD) levels, and suppress the level of malondialdehyde (MDA) in COPD model mice. Moreover, SchA treatment suppressed the generation of the NLRP3/ASC/Caspase1 inflammasome complex to inhibit the inflammatory response caused by IL-1ß and IL-18 and pyroptosis caused by GSDMD. In conclusion, our study shows that SchA treatment can inhibit the production of ROS and the activation of the NLRP3 inflammasome by upregulating Nrf-2, thereby producing anti-inflammatory effects and reducing lung injury in COPD model mice. More importantly, SchA exhibited similar anti-inflammatory effects to dexamethasone in COPD model mice, and we did not observe substantial side effects of SchA treatment. The high safety of SchA makes it a potential candidate drug for the treatment of COPD.

Research Progress on Exosomes in Osteonecrosis of the Femoral Head.

Osteonecrosis of the femoral head (ONFH) is a progressive disease that often necessitates hip replacement if hip preservation therapy fails. ONFH places a heavy economic burden and severe psychological pressure on patients. At present, ONFH is treated by either surgical or non-surgical methods. In clinical practice, stem cells combined with surgery has achieved some positive results, but many problems remain to be resolved. Exosomes are small vesicles of 30-150 nm, which are rich in various nucleic acids, proteins, and small molecules depending on the cells from which they are derived. A growing number of studies have found that exosomes play an important role in tissue damage repair. In comparison with stem cells, exosomes have lower immunogenicity. Also, exosomes can promote cell proliferation and inhibit tumor growth. In addition, exosomes can also be used as natural carriers of drugs. Many studies have shown that exosomes have therapeutic effects in hormone-induced ONFH. Exosomes have the effect of promoting vascular regeneration and show good application prospects in ONFH. Here, we present a review of studies on the application of exosomes in ONFH to provide a reference for future research.

Therapeutic effect of human umbilical cord mesenchymal stem cells in early traumatic osteonecrosis of the femoral head.

Background: Osteonecrosis of the femoral head (ONFH) is a refractory disease due to its unclear pathomechanism. Therapies during the early stage of ONFH have not achieved satisfactory results. Therefore, this study aims to explore the available evidence for the therapeutic effect of human umbilical cord mesenchymal stem cells (HUCMSCs) on early-stage traumatic ONFH. Methods: Early-stage traumatic ONFH was established. The femoral heads of rats were then locally administered HUCMSCs. Four weeks and eight weeks after surgery, bone repair of the necrotic area in the femoral head was analyzed to evaluate the therapeutic effect of HUCMSCs using micro-CT, histopathological staining, immunofluorescence staining, Luminex. Results: HUCMSCs were still present in the femoral head four weeks later, and the morphological, micro-CT and histopathological outcomes in the 4-week HUCMSC-treated group were better than those in the model, NS and 8-week HUCMSC-treated groups. Local transplantation of HUCMSCs promoted bone repair and prevented bone loss in the necrotic area of the femoral head. Conclusions: HUCMSCs can survive and positively affect the femoral head through local transplantation in early-stage traumatic ONFH. The conclusions of this study can provide a treatment option for patients who have ONFH and can serve as basic research on the advanced development of this disease. The Translational potential of this article: The study indicated that the positive effect of exogenous HUCMSCs in the treatment of early-stage traumatic ONFH provides the solid basis and guidance for the clinical application of HUCMSCs.

Injectable adipose-derived stem cells-embedded alginate-gelatin microspheres prepared by electrospray for cartilage tissue regeneration.

Objective: To prepare adipose-derived stem cells (ADSCs)-embedded alginate-gelatinemicrospheres (Alg-Gel-ADSCs MSs) by electrospray and evaluate their feasibility for cartilage tissue engineering. To observe the efficacy of Alg-Gel-ADSCs MSs in repairing articular cartilage defects in SD rats. Methods: ADSCs were isolated and characterized by performing induced differentiation and flow cytometry assays. Alginate-gelatine microspheres with different gelatine concentrations were manufactured by electrospraying, and the appropriate alginate-gelatine concentration and ratio were determined by evaluating microsphere formation. Alg-Gel-ADSCs MSs were compared with Alg-ADSCs MSs through the induction of chondrogenic differentiation and culture. Their feasibility for cartilage tissue engineering was analysed by performing Live/Dead staining, cell proliferation analysis, toluidine blue staining and a glycosaminoglycan (GAG) content analysis. Alg-Gel-ADSCs MSs were implanted in the cartilage defects of SD rats, and the cartilage repair effect was evaluated at different time points. The evaluation included gross observations and histological evaluations, fluorescence imaging tracking, immunohistochemical staining, microcomputed tomography (micro-CT) and a CatWalk evaluation. Results: The isolated ADSCs showed multidirectional differentiation and were used for cartilage tissue engineering. Using 1.5 w:v% alginate and 0.5 w:v% gelatine (Type B), we successfully prepared nearly spherical microspheres. Compared with alginate microspheres, alginate gel increased the viability of ADSCs and promoted the proliferation and chondrogenesis of ADSCs. In our experiments on knee cartilage defects in SD rats in vivo, the Alg-Gel-ADSCs MSs showed superior cartilage repair in cell resides, histology evaluation, micro-CT imaging and gait analysis. Conclusions: Microspheres composed of 1.5 w:v% alginate-0.5 w:v% gelatine increase the viability of ADSCs and supported their proliferation and deposition of cartilage matrix components. ADSCs embedded in 1.5 w:v% alginate-0.5 w:v% gelatine microspheres show superior repair efficacy and prospective applications in cartilage tissue repair. The translational potential of this article: In this study, injectable adipose-derived stem cells-embedded alginate-gelatin microspheres (Alg-Gel-ADSCs MSs) were prepared by the electrospray . Compared with the traditional alginate microspheres, its support ability for ADSCs is better and shows a better repair effect. This study provides a promising strategy for cartilage tissue regeneration.

Report of the First International Symposium on NUT Carcinoma.

NUT carcinoma is a rare, aggressive cancer defined by rearrangements of the NUTM1 gene. No routinely effective treatments of NUT carcinoma exist, despite harboring a targetable oncoprotein, most commonly BRD4-NUT. The vast majority of cases are fatal. Poor awareness of the disease is a major obstacle to progress in the treatment of NUT carcinoma. While the incidence likely exceeds that of Ewing sarcoma, and BRD4-NUT heralded the bromodomain and extra-terminal domain (BET) inhibitor class of selective epigenetic modulators, NUT carcinoma is incorrectly perceived as "impossibly rare," and therefore receives comparatively little private or governmental funding or prioritization by pharma. To raise awareness, propagate scientific knowledge, and initiate a consensus on standard and targeted treatment of NUT carcinoma, we held the First International Symposium on NUT Carcinoma on March 3, 2021. This virtual event had more than eighty attendees from the Americas, Europe, Asia, and Australia. Patients with NUT carcinoma and family members were represented and shared perspectives. Broadly, the four areas discussed by experts in the field included (1) the biology of NUT carcinoma; (2) standard approaches to the treatment of NUT carcinoma; (3) results of clinical trials using BET inhibitors; and (4) future directions, including novel BET bromodomain inhibitors, combinatorial approaches, and immunotherapy. It was concluded that standard chemotherapeutic approaches and first-generation BET bromodomain inhibitors, the latter complicated by a narrow therapeutic window, are only modestly effective in a minority of cases. Nonetheless, emerging second-generation targeted inhibitors, novel rational synergistic combinations, and the incorporation of immuno-oncology approaches hold promise to improve the prognosis of this disease.

Potential and recent advances of microcarriers in repairing cartilage defects.

Articular cartilage regeneration is one of the challenges faced by orthopedic surgeons. Microcarrier applications have made great advances in cartilage tissue engineering in recent years and enable cost-effective cell expansion, thus providing permissive microenvironments for cells. In addition, microcarriers can be loaded with proteins, factors, and drugs for cartilage regeneration. Some microcarriers also have the advantages of injectability and targeted delivery. The application of microcarriers with these characteristics can overcome the limitations of traditional methods and provide additional advantages. In terms of the transformation potential, microcarriers have not only many advantages, such as providing sufficient and beneficial cells, factors, drugs, and microenvironments for cartilage regeneration, but also many application characteristics; for example, they can be injected to reduce invasiveness, transplanted after microtissue formation to increase efficiency, or combined with other stents to improve mechanical properties. Therefore, this technology has enormous potential for clinical transformation. In this review, we focus on recent advances in microcarriers for cartilage regeneration. We compare the characteristics of microcarriers with other methods for repairing cartilage defects, provide an overview of the advantages of microcarriers, discuss the potential of microcarrier systems, and present an outlook for future development. TRANSLATIONAL POTENTIAL OF THIS ARTICLE: We reviewed the advantages and recent advances of microcarriers for cartilage regeneration. This review could give many scholars a better understanding of microcarriers, which can provide doctors with potential methods for treating patients with cartilage injure.

Local administration of zoledronic acid prevents traumatic osteonecrosis of the femoral head in rat model.

BACKGROUND: Osteonecrosis of the femoral head (ONFH) is a refractory disease due to its unclear pathomechanism. Neither conservative treatment nor surgical treatment during the early stage of ONFH achieves satisfactory results. Therefore, this study aims to explore the available evidence on the effect of zoledronic acid on early-stage ONFH. METHODS: For groups were established:the Normal group, model group, Normal saline group(NS group) and zoledronic acid-treated group. The blood supply to the femoral head of animals in the model group and zoledronic acid-treated group was interrupted via a surgical procedure, and zoledronic acid was then locally administered to the femoral head. Four weeks after surgery, all the hips were harvested and evaluated by micro-CT and histopathology(H&E staining, TRAP staining, Toluidine blue staining and masson staining). RESULTS: The values of BMD, BS/BV and Tb.Th in the Normal group and zoledronic acid-treated group were significantly higher than those in the model group and NS group (p â€‹< â€‹0.05). The outcome of H&E staining, Toluidine blue staining and masson staining were consistent with that of micro-CT. CONCLUSION: The local administration of zoledronic acid in the femoral head had positive effects on the bone structure of the femoral head in a modified rat model of traumatic ONFH and offered a promising therapeutic strategy during the early stage of ONFH. THE TRANSLATIONAL POTENTIAL OF THIS ARTICLE: This article could provide a choice for treating patients who have osteonecrosis of femora head and can be the basic research for advanced development over this disease.

The dual PI3K/mTOR inhibitor NVP-BEZ235 prevents epithelial-mesenchymal transition induced by hypoxia and TGF-ß1.

Epithelial-mesenchymal transition (EMT) is regarded as the most important mechanism behind the initiation of cancer metastasis. Though there has been great interest in developing therapies aimed at impairing the process of EMT, only few molecules have been identified to orchestrate it so far. Here we report that the dual PI3K/mTOR inhibitor NVP-BEZ235 is capable of preventing human ovarian cancer cell line SKOV-3 and prostatic cancer cell line PC-3 from hypoxia- and TGF-ß1-induced EMT. The addition of NVP-BEZ235 reverses the EMT-like morphologic changes, down-regulation of E-cadherin, and enhancement of cell migration induced by 1% O2 partially through interfering with the expression and transcriptional activity of Hif-1α via PI3K/mTOR pathway. In addition, NVP-BEZ235 inhibits TGF-ß1-induced phosphorylation of Smad2/3 and Akt/GSK-3ß, reduces the expression of Snail both in transcriptional and post-translational level, and consequently prevents the repression of E-cadherin expression as well as the increase of cell motility caused by TGF-ß1. Moreover, in nude mice bearing SKOV-3 ovarian cancer xenografts, NVP-BEZ235 significantly increases the mRNA level of E-cadherin. Taken together, our study demonstrates, for the first time, NVP-BEZ235 can prevent microenvironment and growth factor induced EMT, which suggests this agent as a potential candidate for cancer metastasis treatment.

DJ-1 mediates the resistance of cancer cells to dihydroartemisinin through reactive oxygen species removal.

Dihydroartemisinin (DHA), one of the main metabolites of artemisinin and its derivatives, presents anti-cancer potential in vitro and in vivo. To explore the mechanisms of resistance toward DHA, a DHA-resistant cell line, HeLa/DHA, was established with a resistance factor of 7.26 in vitro. Upon DHA treatment, apoptotic cells were significantly elicited in parental HeLa cells but minimally induced in HeLa/DHA cells. HeLa/DHA cells also displayed much less sensitivity to DHA-induced tumor suppression in cancer xenograft models than HeLa cells. Intriguingly, DHA-resistant cells did not display a multidrug-resistant phenotype. Based on a proteomic study employing LC-ESI-MS/MS together with pathway analysis, DJ-1 (PARK7) was found to be highly expressed in HeLa/DHA cells. Western blot and immunofluorescence assays confirmed the higher expression of DJ-1 in HeLa/DHA cells than in parental cells in both cell line and xenograft models. DJ-1 is translocated to the mitochondria of HeLa/DHA cells and oxidized, providing DJ-1 with stronger cytoprotection activity. Further study revealed that DJ-1 knockdown in HeLa/DHA cells abolished the observed resistance, whereas overexpression of DJ-1 endowed the parental HeLa cells with resistance toward DHA. Reactive oxygen species (ROS) were also significantly induced by either DHA or hydrogen peroxide in HeLa cells but not in resistant HeLa/DHA cells. When the cells were pretreated with N-acetyl-l-cysteine, the effect of DJ-1 knockdown on sensitizing HeLa/DHA cells to DHA was significantly attenuated. In summary, our study suggests that overexpression and mitochondrial translocation of DJ-1 provides HeLa/DHA cells with resistance to DHA-induced ROS and apoptosis.

GL3, a Novel 4ß-Anilino-4'-O-Demethyl-4-Desoxypodophyllotoxin Analog, Traps Topoisomerase II Cleavage Complexes and Exerts Anticancer Activities.

A novel VP-16 derivative, 4ß-[N -(4‴-acetyloxyl-phenyl-1‴-carbonyl)-4″-aminoanilino]-4'-O-demethyl-4-desoxypodophyllotoxin (GL3), displayed a wide range of cytotoxicity in a panel of human tumor cell lines, with half-maximal inhibitory concentration (IC(50)) values ranging from 0.82 to 4.88 µM, much less than that of VP-16 (4.18-39.43 µM). Importantly, GL3 induces more significant apoptosis and cell cycle arrest than VP-16. The molecular and cellular machinery studies showed that GL3 functions as a topoisomerase II (Top 2) poison through direct binding to the enzyme, and the advanced cell-killing activities of GL3 were ascribed to its potent effects on trapping Top 2-DNA cleavage complex, Moreover, GL3-triggered DNA double-strand breaks and apoptotic cell death were in a Top 2-dependent manner, because the catalytic inhibitor aclarubicin attenuated these biologic consequences caused by Top 2 poisoning in GL3-treated cells. Taken together, among a series of 4ß-anilino-4'-O-demethyl-4-desoxypodophyllotoxin analog, GL3 stood out by its improved anticancer activity and well-defined Top 2 poisoning mechanisms, which merited the potential value of GL3 as an anticancer lead compound/drug candidate deserving further development.

Upregulating Noxa by ER stress, celastrol exerts synergistic anti-cancer activity in combination with ABT-737 in human hepatocellular carcinoma cells.

The human hepatocellular carcinoma (HCC) represents biologically aggressive and chemo-resistant cancers. Owing to the low affinity with the apoptotic factor Mcl-1, the BH3 mimetic drug ABT-737 failed to exert potent cancer-killing activities in variety of cancer models including HCC. The current study demonstrated that combining ABT-737 and Celastrol synergistically suppressed HCC cell proliferation, and induced apoptosis which was accompanied with the activation of caspase cascade and release of cytochrome c from mitochondria. Further study revealed that the enhanced Noxa caused by Celastrol was the key factor for the synergy, since small interfering RNA-mediated knockdown of Noxa expression in HCC cells resulted in decreased apoptosis and attenuated anti-proliferative effects of the combination. In addition, our study unraveled that, upon Celastrol exposure, the activation of endoplasmic reticulum (ER) stress, specifically, the eIF2α-ATF4 pathway played indispensable roles in the activation of Noxa, which was validated by the observation that depletion of ATF4 significantly abrogated the Noxa elevation by Celastrol. Our findings highlight a novel signaling pathway through which Celastrol increase Noxa expression, and suggest the potential use of ATF4-mediated regulation of Noxa as a promising strategy to improve the anti-cancer activities of ABT-737.