Vaccinia viral A26 protein is a fusion suppressor of mature virus and triggers membrane fusion through conformational change at low pH.

Vaccinia mature virus requires A26 envelope protein to mediate acid-dependent endocytosis into HeLa cells in which we hypothesized that A26 protein functions as an acid-sensitive membrane fusion suppressor. Here, we provide evidence showing that N-terminal domain (aa1-75) of A26 protein is an acid-sensitive region that regulates membrane fusion. Crystal structure of A26 protein revealed that His48 and His53 are in close contact with Lys47, Arg57, His314 and Arg312, suggesting that at low pH these His-cation pairs could initiate conformational changes through protonation of His48 and His53 and subsequent electrostatic repulsion. All the A26 mutant mature viruses that interrupted His-cation pair interactions of His48 and His 53 indeed have lost virion infectivity. Isolation of revertant viruses revealed that second site mutations caused frame shifts and premature termination of A26 protein such that reverent viruses regained cell entry through plasma membrane fusion. Together, we conclude that viral A26 protein functions as an acid-sensitive fusion suppressor during vaccinia mature virus endocytosis.

Interleukin-37 is highly expressed in regulatory T cells of melanoma patients and enhanced by melanoma cell secretome.

Immune suppression is one of the 10 hallmarks of cancer. Interleukin-37 (IL-37), a member of the IL-1 family, inhibits both innate and adaptive immunity, and has been shown to modulate immune responses in various disease conditions. Yet, IL-37 has rarely been investigated in cancer patients, and its biological role in cancer remains to be elucidated. In this study, we investigated the gene expression of IL-37 in age- and sex-matched blood samples of healthy individuals and melanoma patients, and demonstrated upregulation of IL-37 messenger RNA (mRNA) in the blood samples of melanoma patients. By further analyzing immune cell subsets responsible for the upregulated IL-37 expression, we discovered that IL-37 mRNA was highly expressed in T cells and granulocytes, with the highest expression in regulatory T (Treg ) cells in healthy individuals, and that IL-37 mRNA was upregulated in lymphocytes (T, B, and natural killer cells) in melanoma patient blood. Among all cell subsets, Treg cells from melanoma patients exhibited the highest IL-37 gene expression levels. We provided evidence that melanoma-conditioned media induces IL-37 mRNA and protein expression in multiple lymphocyte populations, particularly in Treg cells. We further confirmed that the IL-1-mediated secretome from human melanoma cells, specifically transforming growth factor-ß, induces IL-37 mRNA expression in human Treg cells. Our results suggest a potential immunosuppressive role for IL-1 and IL-37 in melanoma tumorigenesis. Highly elevated IL-37 in specific lymphocyte populations could serve as a biomarker for tumor-induced immunosuppression.

Extracellular forms of IL-37 inhibit innate inflammation in vitro and in vivo but require the IL-1 family decoy receptor IL-1R8.

Similar to IL-1α and IL-33, IL-1 family member IL-37b translocates to the nucleus and is associated with suppression of innate and adaptive immunity. Here we demonstrate an extracellular function of the IL-37 precursor and a processed form. Recombinant IL-37 precursor reduced LPS-induced IL-6 by 50% (P < 0.001) in highly inflammatory human blood-derived M1 differentiated macrophages derived from selective subjects but not M2 macrophages. In contrast, a neutralizing monoclonal anti-IL-37 increased LPS-induced IL-6, TNFα and IL-1ß (P < 0.01). The suppression by IL-37 was consistently observed at low picomolar but not nanomolar concentrations. Whereas LPS induced a 12-fold increase in TNFα mRNA, IL-37 pretreatment decreased the expression to only 3-fold over background (P < 0.01). Mechanistically, LPS-induced p38 and pERK were reduced by IL-37. Recombinant IL-37 bound to the immobilized ligand binding α-chain of the IL-18 receptor as well as to the decoy receptor IL-1R8. In M1 macrophages, LPS increased the surface expression of IL-1R8. Compared with human blood monocytes, resting M1 cells express more surface IL-1R8 as well as total IL-1R8; there was a 16-fold increase in IL-1R8 mRNA levels when pretreated with IL-37. IL-37 reduced LPS-induced TNFα and IL-6 by 50-55% in mouse bone marrow-derived dendritic cells, but not in dendritic cells derived from IL-1R8-deficient mice. In mice subjected to systemic LPS-induced inflammation, pretreatment with IL-37 reduced circulating and organ cytokine levels. Thus, in addition to a nuclear function, IL-37 acts as an extracellular cytokine by binding to the IL-18 receptor but using the IL-1R8 for its anti-inflammatory properties.

Downregulation of CITED2 contributes to TGFß-mediated senescence of tendon-derived stem cells.

Tendon-derived stem cells (TDSCs) are multipotent adult stem cells with potential applications in tendon and tendon-bone junction repair. However, cellular characteristics change during in vitro passaging. Therefore, elucidation of the molecular and cellular mechanisms of tendon aging will be essential for the development of TDSC-based therapies. The aim of this study is to investigate the effect of CITED2, a nuclear regulator and transforming growth factor ß2 (TGFß2) on TDSC proliferation and senescence by comparing cells derived from Achilles tendon biopsies of young individuals (Y-TDSC) with those of older patients (O-TDSC). Our results showed that CITED2 mRNA and protein expression levels were significantly higher in Y-TDSCs than in O-TDSCs and O-TDSCs displayed decreased proliferation and increased senescence compared with Y-TDSCs. Furthermore, high levels of CITED2 protein expression in Y-TDSCs correlated with the downregulation of SP1 and p21 and the upregulation of MYC, potentially indicating the mechanism by which CITED2 upregulates TDSC proliferation. TGFß2 was found to downregulate the expression of the CITED2 gene and knockdown of CITED2 abolished the effect of TGFß2 on TDSC proliferation and senescence. Thus, the downregulation of CITED2 contributes to TGFß-mediated senescence providing an insight into the molecular and cellular mechanisms that contribute to tendon aging and degeneration. Our findings may aid the development of cell-based therapies for tendon repair.

Suppression of antigen-specific adaptive immunity by IL-37 via induction of tolerogenic dendritic cells.

IL-1 family member IL-37 limits innate inflammation in models of colitis and LPS-induced shock, but a role in adaptive immunity remains unknown. Here, we studied mice expressing human IL-37b isoform (IL-37tg) subjected to skin contact hypersensitivity (CHS) to dinitrofluorobenzene. CHS challenge to the hapten was significantly decreased in IL-37tg mice compared with wild-type (WT) mice (-61%; P < 0.001 at 48 h). Skin dendritic cells (DCs) were present and migrated to lymph nodes after antigen uptake in IL-37tg mice. When hapten-sensitized DCs were adoptively transferred to WT mice, antigen challenge was greatly impaired in mice receiving DCs from IL-37tg mice compared with those receiving DCs from WT mice (-60%; P < 0.01 at 48 h). In DCs isolated from IL-37tg mice, LPS-induced increase of MHC II and costimulatory molecule CD40 was reduced by 51 and 31%, respectively. In these DCs, release of IL-1ß, IL-6, and IL-12 was reduced whereas IL-10 secretion increased (37%). Consistent with these findings, DCs from IL-37tg mice exhibited a lower ability to stimulate syngeneic and allogeneic naive T cells as well as antigen-specific T cells and displayed enhanced induction of T regulatory (Treg) cells (86%; P < 0.001) in vitro. Histological analysis of CHS skin in mice receiving hapten-sensitized DCs from IL-37tg mice revealed a marked reduction in CD8(+) T cells (-74%) but an increase in Treg cells (2.6-fold). Together, these findings reveal that DCs expressing IL-37 are tolerogenic, thereby impairing activation of effector T-cell responses and inducing Treg cells. IL-37 thus emerges as an inhibitor of adaptive immunity.

MiR397b regulates both lignin content and seed number in Arabidopsis via modulating a laccase involved in lignin biosynthesis.

Plant laccase (LAC) enzymes belong to the blue copper oxidase family and polymerize monolignols into lignin. Recent studies have established the involvement of microRNAs in this process; however, physiological functions and regulation of plant laccases remain poorly understood. Here, we show that a laccase gene, LAC4, regulated by a microRNA, miR397b, controls both lignin biosynthesis and seed yield in Arabidopsis. In transgenic plants, overexpression of miR397b (OXmiR397b) reduced lignin deposition. The secondary wall thickness of vessels and the fibres was reduced in the OXmiR397b line, and both syringyl and guaiacyl subunits are decreased, leading to weakening of vascular tissues. In contrast, overexpression of miR397b-resistant laccase mRNA results in an opposite phenotype. Plants overexpressing miR397b develop more than two inflorescence shoots and have an increased silique number and silique length, resulting in higher seed numbers. In addition, enlarged seeds and more seeds are formed in these miR397b overexpression plants. The study suggests that miR397-mediated development via regulating laccase genes might be a common mechanism in flowering plants and that the modulation of laccase by miR397 may be potential for engineering plant biomass production with less lignin.

ALDH1A isozymes are markers of human melanoma stem cells and potential therapeutic targets.

Although the concept of cancer stem cells (CSCs) is well-accepted for many tumors, the existence of such cells in human melanoma has been the subject of debate. In this study, we demonstrate the existence of human melanoma cells that fulfill the criteria for CSCs (self-renewal and differentiation) by serially xenotransplanting cells into nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice. These cells possess high aldehyde dehydrogenase (ALDH) activity with ALDH1A1 and ALDH1A3 being the predominant ALDH isozymes. ALDH-positive melanoma cells are more tumorigenic than ALDH-negative cells in both NOD/SCID mice and NSG mice. Biological analyses of the ALDH-positive melanoma cells reveal the ALDH isozymes to be key molecules regulating the function of these cells. Silencing ALDH1A by siRNA or shRNA leads to cell cycle arrest, apoptosis, decreased cell viability in vitro, and reduced tumorigenesis in vivo. ALDH-positive melanoma cells are more resistant to chemotherapeutic agents and silencing ALDH1A by siRNA sensitizes melanoma cells to drug-induced cell death. Furthermore, we, for the first time, examined the molecular signatures of ALDH-positive CSCs from patient-derived tumor specimens. The signatures of melanoma CSCs include retinoic acid (RA)-driven target genes with RA response elements and genes associated with stem cell function. These findings implicate that ALDH isozymes are not only biomarkers of CSCs but also attractive therapeutic targets for human melanoma. Further investigation of these isozymes and genes will enhance our understanding of the molecular mechanisms governing CSCs and reveal new molecular targets for therapeutic intervention of cancer.

Constitutively active inflammasome in human melanoma cells mediating autoinflammation via caspase-1 processing and secretion of interleukin-1beta.

Interleukin-1beta (IL-1beta) is a pleiotropic cytokine promoting inflammation, angiogenesis, and tissue remodeling as well as regulation of immune responses. Although IL-1beta contributes to growth and metastatic spread in experimental and human cancers, the molecular mechanisms regulating the conversion of the inactive IL-1beta precursor to a secreted and active cytokine remains unclear. Here we demonstrate that NALP3 inflammasome is constitutively assembled and activated with cleavage of caspase-1 in human melanoma cells. Late stage human melanoma cells spontaneously secrete active IL-1beta via constitutive activation of the NALP3 inflammasome and IL-1 receptor signaling, exhibiting a feature of autoinflammatory diseases. Unlike human blood monocytes, these melanoma cells require no exogenous stimulation. In contrast, NALP3 functionality in intermediate stage melanoma cells requires activation of the IL-1 receptor to secrete active IL-1beta; cells from an early stage of melanoma require stimulation of the IL-1 receptor plus the co-stimulant muramyl dipeptide. The spontaneous secretion of IL-1beta from melanoma cells was reduced by inhibition of caspase-1 or the use of small interfering RNA directed against ASC. Supernatants from melanoma cell cultures enhanced macrophage chemotaxis and promoted in vitro angiogenesis, both prevented by pretreating melanoma cells with inhibitors of caspases-1 and -5 or IL-1 receptor blockade. These findings implicate IL-1-mediated autoinflammation as contributing to the development and progression of human melanoma and suggest that inhibiting the inflammasome pathway or reducing IL-1 activity can be a therapeutic option for melanoma patients.

Green tea polyphenol epigallocatechin-3-gallate suppresses melanoma growth by inhibiting inflammasome and IL-1ß secretion.

Epigallocatechin-3-gallate (EGCG), the major polyphenolic component of green tea, has been demonstrated to possess anti-inflammatory, antioxidant, anti-mutagenic and anti-carcinogenic properties. The anti-melanoma effect of EGCG has been previously suggested, but no clear mechanism of action has been established. In this study, we demonstrated that EGCG inhibits melanoma cell growth at physiological doses (0.1-1 µM). In the search for mechanisms of EGCG-mediated melanoma cell suppression, we found that NF-κB was inhibited, and that reduced NF-κB activity was associated with decreased IL-1ß secretion from melanoma cells. Since inflammasomes are involved in IL-1ß secretion, we investigated whether IL-1ß suppression was mediated by inflammasomes, and found that EGCG treatment led to downregulation of the inflammasome component, NLRP1, and reduced caspase-1 activation. Furthermore, silencing the expression of NLRP1 abolished EGCG-induced inhibition of tumor cell proliferation both in vitro and in vivo, suggesting a key role of inflammasomes in EGCG efficacy. This paper provides a novel mechanism for EGCG-induced melanoma inhibition: inflammasome downregulation→decreased IL-1ß secretion→decreased NF-κB activities→decreased cell growth. In addition, it suggests inflammasomes and IL-1ß could be potential targets for future melanoma therapeutics.

IL-3 induces basophil expansion in vivo by directing granulocyte-monocyte progenitors to differentiate into basophil lineage-restricted progenitors in the bone marrow and by increasing the number of basophil/mast cell progenitors in the spleen.

Recent work has established important roles for basophils in regulating immune responses. To exert their biological functions, basophils need to be expanded to critical numbers. However, the mechanisms underlying basophil expansion remain unclear. In this study, we established that IL-3 played an important role in the rapid and specific expansion of basophils. We found that the IL-3 complex (IL-3 plus anti-IL-3 Ab) greatly facilitated the differentiation of GMPs into basophil lineage-restricted progenitors (BaPs) but not into eosinophil lineage-restricted progenitors or mast cells in the bone marrow. We also found that the IL-3 complex treatment resulted in approximately 4-fold increase in the number of basophil/mast cell progenitors (BMCPs) in the spleen. IL-3-driven basophil expansion depended on STAT5 signaling. We showed that GMPs but not common myeloid progenitors expressed low levels of IL-3 receptor. IL-3 receptor expression was dramatically up-regulated in BaPs but not eosinophil lineage-restricted progenitors. Approximately 38% of BMCPs expressed the IL-3R alpha-chain. The up-regulated IL-3 receptor expression was not affected by IL-3 or STAT5. Our findings demonstrate that IL-3 induced specific expansion of basophils by directing GMPs to differentiate into BaPs in the bone marrow and by increasing the number of BMCPs in the spleen.

Roles of cysteines Cys115 and Cys201 in the assembly and thermostability of grouper betanodavirus particles.

The virus-like particle (VLP) assembled from capsid subunits of the dragon grouper nervous necrosis virus (DGNNV) is very similar to its native T = 3 virion. In order to investigate the effects of four cysteine residues in the capsid polypeptide on the assembly/dissociation pathways of DGNNV virions, we recombinantly cloned mutant VLPs by mutating each cysteine to destroy the specific disulfide linkage as compared with thiol reduction to destroy all S-S bonds. The mutant VLPs of C187A and C331A mutations were similar to wild-type VLPs (WT-VLPs); hence, the effects of Cys187 and Cys331 on the particle formation and thermostability were presumably negligible. Electron microscopy showed that either C115A or C201A mutation disrupted de novo VLP formation significantly. As shown in micrographs and thermal decay curves, beta-mercaptoethanol-treated WT-VLPs remained intact, merely resulting in lower tolerance to thermal disruption than native WT-VLPs. This thiol reduction broke disulfide linkages inside the pre-fabricated VLPs, but it did not disrupt the appearance of icosahedrons. Small dissociated capsomers from EGTA-treated VLPs were able to reassemble back to icosahedrons in the presence of calcium ions, but additional treatment with beta-mercaptoethanol during EGTA dissociation resulted in inability of the capsomers to reassemble into the icosahedral form. These results indicated that Cys115 and Cys201 were essential for capsid formation of DGNNV icosahedron structure in de novo assembly and reassembly pathways, as well as for the thermal stability of pre-fabricated particles.

CDK1 Interacts with Sox2 and Promotes Tumor Initiation in Human Melanoma.

: Cancers are composed of heterogeneous subpopulations with various tumor-initiating capacities, yet key stem cell genes associated with enhanced tumor-initiating capacities and their regulatory mechanisms remain elusive. Here, we analyzed patient-derived xenografts from melanoma, colon, and pancreatic cancer tissues and identified enrichment of tumor-initiating cells in MHC class I-hi cells, where CDK1, a master regulator of the cell cycle, was upregulated. Overexpression of CDK1, but not its kinase-dead variant, in melanoma cells increased their spheroid forming ability, tumorigenic potential, and tumor-initiating capacity; inhibition of CDK1 with pharmacologic agents reduced these characteristics, which was unexplained by the role of CDK1 in regulating the cell cycle. Proteomic analysis revealed an interaction between CDK1 and the pluripotent stem cell transcription factor Sox2. Blockade or knockdown of CDK1 resulted in reduced phosphorylation, nuclear localization, and transcriptional activity of Sox2. Knockout of Sox2 in CDK1-overexpressing cells reduced CDK1-driven tumor-initiating capacity substantially. Furthermore, GSEA analysis of CDK1hi tumor cells identified a pathway signature common in all three cancer types, including E2F, G2M, MYC, and spermatogenesis, confirming a stem-like nature of CDK1hi tumor cells. These findings reveal a previously unrecognized role for CDK1 in regulating tumor-initiating capacity in melanoma and suggest a novel treatment strategy in cancer via interruption of CDK1 function and its protein-protein interactions. SIGNIFICANCE: These findings uncover CDK1 as a new regulator of Sox2 during tumor initiation and implicate the CDK1-Sox2 interaction as a potential therapeutic target in cancer.

Use of a MCL-1 inhibitor alone to de-bulk melanoma and in combination to kill melanoma initiating cells.

MCL-1 (BCL-2 family anti-apoptotic protein) is responsible for melanoma's resistance to therapy. Cancer initiating cells also contribute to resistance and relapse from treatments. Here we examined the effects of the MCL-1 inhibitor SC-2001 in killing non melanoma-initiating-cells (bulk of melanoma), and melanoma-initiating-cells (MICs). By itself, SC-2001 significantly kills melanoma cells under monolayer conditions in vitro and in a conventional mouse xenograft model. However, even at high doses (10µM), SC-2001 does not effectively eliminate MICs. In contrast, the combination of SC-2001 with ABT-737 (a BCL-2/BCL-XL/BCL-W inhibitor) significantly decreases ALDH+ cells, disrupts primary spheres, and inhibits the self-renewability of MICs. These results were observed in multiple melanomas, including short term cultures of relapsed tumors from current treatments, independent of the mutation status of BRAF or NRAS. Using a low-cell-number mouse xenograft model, we examined the effects of these treatments on the tumor initiating ability of MIC-enriched cultures. The combination therapy reduces tumor formation significantly compared to either drug alone. Mechanistic studies using shRNA and the CRISPR-Cas9 technology demonstrated that the upregulation of pro-apoptotic proteins NOXA and BIM contribute to the combination-induced cell death. These results indicate that the MCL-1 inhibitor SC-2001 combined with ABT-737 is a promising treatment strategy for targeting melanoma.

Rice embryogenic calli express a unique set of microRNAs, suggesting regulatory roles of microRNAs in plant post-embryogenic development.

In vitro cultured embryogenic callus was employed as a model to investigate microRNAs (miRNAs) associated with embryogenesis and post-embryonic development. Thirty-one miRNAs including 16 novel species were identified from a large number of small RNAs which were cloned from both differentiated and undifferentiated rice embryogenic calli. Four target genes of the miRNAs were further validated. A set of the miRNAs, including miR397 and miR156, exhibited intriguing expression patterns during the transition from undifferentiated to differentiated calli. By exploiting the correlations between the differential expression patterns of these miRNAs and their targets, the regulatory roles of the miRNAs on meristem maintenance and embryogenesis were indicated.

Combining a GSI and BCL-2 inhibitor to overcome melanoma's resistance to current treatments.

Major limitations of current melanoma treatments are for instances of relapse and the lack of therapeutic options for BRAF wild-type patients who do not respond to immunotherapy. Many studies therefore focus on killing resistant subpopulations, such as Melanoma Initiating Cells (MICs) to prevent relapse. Here we examined whether combining a GSI (γ-Secretase Inhibitor) with ABT-737 (a small molecule BCL-2/BCL-XL/BCL-W inhibitor) can kill both the non-MICs (bulk of melanoma) and MICs. To address the limitations of melanoma therapies, we included multiple tumor samples of patients relapsed from current treatments, with a diverse genetic background (with or without the common BRAF, NRAS or NF1 mutations) in these studies. Excitingly, the combination treatment reduced cell viability and induced apoptosis of the non-MICs; disrupted primary spheres, decreased the ALDH+ cells, and inhibited the self-renewability of the MICs in multiple melanoma cell lines and relapsed patient samples. Using a low-cell-number mouse xenograft model, we demonstrated that the combination significantly reduced the tumor initiating ability of MIC-enriched cultures from relapsed patient samples. Mechanistic studies also indicate that cell death is NOXA-dependent. In summary, this combination may be a promising strategy to address treatment relapse and for triple wild-type patients who do not respond to immunotherapy.

Understanding melanoma stem cells.

Tumors are incredibly diverse and contain many different subpopulations of cells. The cancer stem cell (CSC) subpopulation is responsible for many aspects of tumorigenesis and has been shown to play an important role in melanoma development, progression, drug resistance and metastasis. However, it is becoming clear that tumor cell populations are dynamic and can be influenced by many factors, such as signals from the tumor microenvironment and somatic evolution. This review will present the current understanding of CSCs and the challenges of identifying and characterizing this dynamic cell population. The known characteristics and functions of melanoma stem cells, and the potential for therapeutic targeting of these cells in melanoma, will be discussed.

Efficacy of tripterygium glycosides tablet in treating ankylosing spondylitis: a systematic review and meta-analysis of randomized controlled trials.

Hundreds of randomized controlled trials (RCTs) on tripterygium glycosides tablet (TGT) in the treatment of ankylosing spondylitis (AS) have been published, but the therapeutic effects have never been systematically reviewed yet. The aim of this meta-analysis was to evaluate the efficacy of TGT on AS based on RCTs. PubMed, ScienceDirect, Cochrane Library, China Journals Full-text Database, and Wanfang Data were searched. The RCT quality was evaluated by the Cochrane Collaboration's tool for assessing risk of bias. The RCT characteristics including publication years, sample sizes, and follow-up periods as well as outcome measures including symptoms improvement, morning stiffness (MS), bath ankylosing spondylitis patient global score (BAS-G), pain index (PI), swelling index (SI), finger to floor distance (FFD), pillow wall distance (PWD), Schober test (Schober), erythrocyte sedimentation rate (ESR), and C reactive protein (CRP) were extracted. The odds ratio (OR), mean difference (MD), and its 95% confidence interval (CI) were selected for overall effect sizes. Subgroup, sensitivity, and meta-regression analyses were conducted to confirm the results. Eleven RCTs with 807 participants were included, the quality of which was moderate. OR of TGT in treating AS was 0.46 (95% CI 0.24, 0.90]. MD of MS was 11.79 (95% CI 3.13, 20.45). MD of BAS-G was 0.13 (95% CI -19.73, 19.99). MD of PI was 0.78 (95% CI 0.22, 1.34). MD of SI was 0.80 (95% CI 0.06, 1.53). MD of FFD was 0.80 (95% CI 0.06, 1.53). MD of PWD was 1.37 (95% CI -0.64, 3.38). MD of Schober was -0.36 (95% CI -0.65, -0.07]. MD of ESR was 4.58 (95% CI 2.10, 7.06). MD of CRP was 1.86 (95% CI -2.03, 5.76). Subgroup, sensitivity, and meta-regression analyses found the robust results. In conclusion, TGT could not treat AS effectively, as suggested by the moderate RCT quality and meta-analysis evidence.

Combining a BCL2 inhibitor with the retinoid derivative fenretinide targets melanoma cells including melanoma initiating cells.

Investigations from multiple laboratories support the existence of melanoma initiating cells (MICs) that potentially contribute to melanoma's drug resistance. ABT-737, a small molecule BCL-2/BCL-XL/BCL-W inhibitor, is promising in cancer treatments, but not very effective against melanoma, with the antiapoptotic protein MCL-1 as the main contributor to resistance. The synthetic retinoid fenretinide N-(4-hydroxyphenyl)retinamide (4-HPR) has shown promise for treating breast cancers. Here, we tested whether the combination of ABT-737 with 4-HPR is effective in killing both the bulk of melanoma cells and MICs. The combination synergistically decreased cell viability and caused cell death in multiple melanoma cells lines (carrying either BRAF or NRAS mutations) but not in normal melanocytes. The combination increased the NOXA expression and caspase-dependent MCL-1 degradation. Knocking down NOXA protected cells from combination-induced apoptosis, implicating the role of NOXA in the drug synergy. The combination treatment also disrupted primary spheres (a functional assay for MICs) and decreased the percentage of aldehyde dehydrogenase (high) cells (a marker of MICs) in melanoma cell lines. Moreover, the combination inhibited the self-renewal capacity of MICs, measured by secondary sphere-forming assays. In vivo, the combination inhibited tumor growth. Thus, this combination is a promising treatment strategy for melanoma, regardless of mutation status of BRAF or NRAS.

Targeting glucose metabolism in chondrosarcoma cells enhances the sensitivity to doxorubicin through the inhibition of lactate dehydrogenase-A.

Chondrosarcoma is a malignant cartilage-forming cancer composed of cells derived from transformed cells that produce cartilage. Conventional chemotherapy and radiotherapy have very limited efficacy in patients with advanced chondrosarcoma. In the present study, we reported a novel therapeutic approach in the treatment of chondrosarcoma cells. We detected that lactate dehydrogenase-A (LDHA) is highly active in chondrosarcoma cells and chondrosarcoma patient samples compared with normal chondrocyte cell lines and primary human chondrocyte. Moreover, chondrosarcoma cells exhibited elevated levels of LDHA expression under doxorubicin treatment. To further explore the mechanisms, we generated doxorubicin-resistant cells from SW1353 chondrosarcoma cell line. Notably, the activity and expression of LDHA are upregulated in doxorubicin-resistant cells. Moreover, our data showed a strong correlation between glucose metabolism and doxorubicin resistance in chondrosarcoma cells; doxorubicin-resistant cells displayed highly activated glucose metabolism and depended more on glucose supply. Finally, we reported a synergistic effect produced by incorporating doxorubicin with glycolysis inhibitors-oxamate in the combined treatment of chondrosarcoma cells in vitro and in vivo. In summary, the present study may aid in the development of new approaches using the combination of chemotherapeutic agents for the treatment of chondrosarcoma patients.