Safety and Immunogenicity of the ID93 + GLA-SE Tuberculosis Vaccine in BCG-Vaccinated Healthy Adults: A Randomized, Double-Blind, Placebo-Controlled Phase 2 Trial.

INTRODUCTION: This randomized, double-blind, placebo-controlled, phase 2a trial was conducted to evaluate the safety and immunogenicity of the ID93 + glucopyranosyl lipid adjuvant (GLA)-stable emulsion (SE) vaccine in human immunodeficiency virus (HIV)-negative, previously Bacillus Calmette-Guérin (BCG)-vaccinated, and QuantiFERON-TB-negative healthy adults in South Korea. METHODS: Adults (n = 107) with no signs or symptoms of tuberculosis were randomly assigned to receive three intramuscular injections of 2 µg ID93 + 5 µg GLA-SE, 10 µg ID93 + 5 µg GLA-SE, or 0.9% normal saline placebo on days 0, 28, and 56. For safety assessment, data on solicited adverse events (AEs), unsolicited AEs, serious AEs (SAEs), and special interest AEs were collected. Antigen-specific antibody responses were measured using serum enzyme-linked immunosorbent assay. T-cell immune responses were measured using enzyme-linked immunospot and intracellular cytokine staining. RESULTS: No SAEs, deaths, or AEs leading to treatment discontinuation were found. The solicited local and systemic AEs observed were consistent with those previously reported. Compared with adults administered with the placebo, those administered with three intramuscular vaccine injections exhibited significantly higher antigen-specific antibody levels and Type 1 T-helper cellular immune responses. CONCLUSION: The ID93 + GLA-SE vaccine induced antigen-specific cellular and humoral immune responses, with an acceptable safety profile in previously healthy, BCG-vaccinated, Mycobacterium tuberculosis-uninfected adult healthcare workers. TRIAL REGISTRATION: This clinical trial was retrospectively registered on 16 January 2019 at Clinicaltrials.gov (NCT03806686).

Effects of pH and NaCl on hydrolysis and transpeptidation activities of a salt-tolerant γ-glutamyltranspeptidase from Bacillus amyloliquefaciens S0904.

Bacillus amyloliquefaciens S0904 was selected as a hyperproducer of a glutamine-hydrolyzing enzyme which was identified as a γ-glutamyltranspeptidase catalyzing both hydrolysis and transpeptidation of glutamyl substrates. The signal peptide-truncated recombinant enzyme (rBAGGT) showed two-fold enhanced specific activity for hydrolysis and optimum pH shift to pH 7 from pH 6 compared with the wild type. The hydrolysis activity of rBAGGT was tolerant against NaCl up to 2.5 M, whereas the transpeptidation activity decreased by NaCl. At pH 6, the addition of 1.5 M NaCl not only enhanced the hydrolysis activity but also inhibited the transpeptidation activity to be ignorable. By contrast, at pH 9 in the absence of NaCl, the alkaline pH-favored transpeptidation activity was 99% of the maximum with only 15% of the maximum hydrolysis activity. In conclusion, the hydrolysis and transpeptidation activities of the recombinant BAGGT is controllable by changing pH and whether or not to add NaCl. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10068-021-00928-6.

Development of a quantitative prediction algorithm for target organ-specific similarity of human pluripotent stem cell-derived organoids and cells.

Human pluripotent stem cell (hPSC)-derived organoids and cells have similar characteristics to human organs and tissues. Thus, in vitro human organoids and cells serve as a superior alternative to conventional cell lines and animal models in drug development and regenerative medicine. For a simple and reproducible analysis of the quality of organoids and cells to compensate for the shortcomings of existing experimental validation studies, a quantitative evaluation method should be developed. Here, using the GTEx database, we construct a quantitative calculation system to assess similarity to the human organs. To evaluate our system, we generate hPSC-derived organoids and cells, and detected organ similarity. To facilitate the access of our system by researchers, we develop a web-based user interface presenting similarity to the appropriate organs as percentages. Thus, this program could provide valuable information for the generation of high-quality organoids and cells and a strategy to guide proper lineage-oriented differentiation.

Impedance Measurement System for Assessing the Barrier Integrity of Three-Dimensional Human Intestinal Organoids.

Human pluripotent stem cell (hPSC)-derived intestinal organoids (HIOs) hold unprecedented promise for basic biology and translational applications. However, developing a quantitative method to evaluate the epithelial cell membrane integrity of HIOs as an in vitro intestinal barrier model is a major challenge because of their complex three-dimensional (3D) structure. In this study, we developed an impedance system to measure the change in electrical resistance of 3D HIOs depending on the integrity of the intestinal epithelial cell membrane, which can reflect functionality and maturity. The expression of intestinal maturation- and tight junction-related markers was significantly higher in HIOs matured in vitro by treatment with IL-2 than in control HIOs. Analysis of gap junction size indicated that mature HIOs have greater integrity, with approximately 30% more compact gaps than immature HIOs. We designed a multi-microchannel system controlled by the inhalation pressure where the HIO is loaded, which enhances the stability and sensitivity of the impedance signal. We demonstrated the applicability of the impedance system by showing the difference in resistance between control and mature HIOs, reflecting the expression of tight junction proteins and their maturation status. We also validated the impedance system by monitoring its resistance in real time during junctional damage to HIOs induced by a digestive agent. In summary, we suggest a quantitative method to directly quantify the physiological changes in complex 3D organoid structures based on impedance spectroscopy, which can be applied to noninvasively monitor live cells and therefore enable their use in subsequent experiments.

TNFα enhances trovafloxacin-induced in vitro hepatotoxicity by inhibiting protective autophagy.

Trovafloxacin (TVX) is associated with idiosyncratic drug-induced liver injury (iDILI) and inflammation-mediated hepatotoxicity. However, the inflammatory stress-regulated mechanisms in iDILI remain unclear. Herein, we elucidated the novel role of tumor-necrosis factor alpha (TNFα), an inflammatory stress factor, in TVX-induced in vitro hepatotoxicity and synergistic toxicity. TVX specifically induced synergistic toxicity in HepG2 cells with TNFα, which inhibits autophagy. TVX-treated HepG2 cells induced protective autophagy by inhibiting the expression of mTOR signaling proteins, while ATG5 knockdown in HepG2 cells, responsible for the impairment of autophagy, enhanced TVX-induced toxicity due to the increase in cytochrome C release and JNK pathway activation. Interestingly, the expression of mTOR signal proteins, which were suppressed by TVX, disrupted the negative feedback of the PI3K/AKT pathway and TNFα rebounded p70S6K phosphorylation. Co-treatment with TVX and TNFα inhibited protective autophagy by maintaining p70S6K activity, which enhanced TVX-induced cytotoxicity. Phosphorylation of p70S6K was inhibited by siRNA knockdown and rapamycin to restore TNFα-inhibited autophagy, which prevented the synergistic effect on TVX-induced cytotoxicity. These results indicate that TVX activates protective autophagy in HepG2 cells exposed to toxicity and an imbalance in negative feedback regulation of autophagy by TNFα synergistically enhanced the toxicity. The finding from this study may contribute to a better understanding of the mechanisms underlying iDILI associated with inflammatory stress.

Quantification of non-alcoholic fatty liver disease progression in 3D liver microtissues using impedance spectroscopy.

Non-alcoholic fatty liver disease (NAFLD) has become a global pandemic. However, a pharmacological cure has not been approved for NAFLD treatment. The greatest barriers to the development of new treatments are the ambiguous criteria among the NAFLD stages and the lack of quantitative methodologies for its disease assessment in a translatable preclinical model. In this study, we developed impedance assessment systems to quantify NAFLD progression in three-dimensional (3D) liver microtissue (hMT). The hMT model undergoing NAFLD represents clinical-like characteristics for a range of stages, such as lipid accumulation, cell ballooning, and stiffening. Each stage can be quantitatively assessed by an impedance system with microchannels under constant or dynamic pressure, depending on the relevant mechanical and morphological changes used in the clinical assessment of NAFLD. We determined a correlation between the impedance parameters and pathophysiological characteristics, such as gap widening and cytoplasmic deformation associated with NAFLD progression using bioimpedance simulation, showing hMTs struggling to return to normal states. In addition, we identified the relative stiffness to assess fibrogenesis from the correlation of resistance change and elongation length into the smaller channel of hMTs. We hope this methodology will have a significant impact on drug development by facilitating improved NAFLD assessment.

TIPRL potentiates survival of lung cancer by inducing autophagy through the eIF2α-ATF4 pathway.

Autophagy, an intracellular system of degrading damaged organelles and misfolded proteins, is essential for cancer cell survival. Despite the progress made towards understanding the mechanism, identification of novel autophagy regulators presents a major obstacle in developing anticancer therapies. Here, we examine the association between the TOR signaling pathway regulator-like (TIPRL) protein and autophagy in malignant transformation of tumors. We show that TIPRL upregulation in non-small cell lung cancer (NSCLC) potentiated autophagy activity and enabled autophagic clearance of metabolic and cellular stress, conferring a survival advantage to cancer cells. Importantly, the interaction of TIPRL with eukaryotic initiation factor 2α (eIF2α) led to eIF2α phosphorylation and activation of the eIF2α-ATF4 pathway, thereby inducing autophagy. Conversely, TIPRL depletion increased apoptosis by reducing autophagic clearance, which was markedly enhanced in TIPRL-depleted A549 xenografts treated with 2-deoxy-D-glucose. Overall, the study indicated that TIPRL is a potential regulator of autophagy and an important drug target for lung cancer therapy.

Upregulation of S100A9 contributes to the acquired resistance to BRAF inhibitors.

BACKGROUNDS: Acquired resistance is a significant clinical challenge in targeted therapy of melanomas using BRAF inhibitors. We previously identified that downregulation of miR-92a-1-5p confers acquired resistance to BRAF inhibition using an miRNA array platform. OBJECTIVE: In this study, we investigated the target genes of miR-92a-1-5p and their functional significance in BRAF inhibitor resistance. METHODS: The miRNA target prediction data were combined with RNA-Seq data to identify possible target genes for miR-92a-1-5p. Cellular effects of target genes were further examined using siRNA knockdown, WST-1 assay, and immunoblotting analysis. RESULTS: We selected S100 calcium-binding protein A9 (S100A9) as a possible target gene for functional validation. S100A9 knockdown abrogated resistance to PLX4720 in A375P/Mdr cells. This result was similar to those described earlier for miR-92a-1-5p, indicating that miR-92a-1-5p inhibits cell viability by targeting S100A9. S100A9 overexpression partially conferred PLX4720 resistance to A375P cells. We also demonstrated that MAPK re-activation does not contribute to the promotion of BRAF inhibitor resistance by S100A9. CONCLUSION: Taken together, our results indicate that S100A9 might be functionally involved in development of resistance to BRAF inhibitors and might be a target for melanoma therapy in the future.

Human three-dimensional in vitro model of hepatic zonation to predict zonal hepatotoxicity.

BACKGROUND: Various hepatic models mimicking liver lobules have been investigated to evaluate the potential hepatotoxic effects of chemicals and drugs, but in vitro hepatic models of zonal hepatotoxicity have not yet been established. Herein, we developed a three-dimensional (3D) hepatic zonal channel to evaluate zone-specific hepatotoxicity. Based on the perivenous zone-3-like cytochrome P450 (CYP) expression patterns in metabolically active HepaRG cells treated with CHIR99021 (CHIR), which is an inducer of Wnt/ß-catenin signaling, this culture model represents a novel tool for exploring hepatic zonation. RESULTS: We generated and validated a 3D hepatic zonal channel model in which 3D HepaRG cells were well distributed in agarose hydrogel channels, and a linear gradient of CHIR was generated according to the zonal distance. According to the results from imaging analyses and bioanalytical experiments, acetaminophen (APAP) caused cytotoxicity in the zone-3 region of the 3D hepatic zonal channel, and the levels of nonphosphorylated ß-catenin, CYP2E, and apoptotic proteins were remarkably increased in the zone-3-like region. Finally, the applicability of the 3D hepatic zonal channel model for the high-throughput screening of zonal hepatotoxicity was successfully evaluated using hepatotoxic drugs, including tamoxifen, bromobenzene, and APAP. CONCLUSIONS: The results indicated that tamoxifen induced cytotoxic effects, regardless of the zonal distance, while the zone-3-specific hepatotoxic drugs bromobenzene and APAP induced greater cytotoxic effects on cells in the zone-3-like region. This finding highlights the potential of our 3D hepatic zonation model as a valuable tool for replicating and evaluating zonal hepatotoxicity by mimicking the spatial features of liver lobules.

Differential Gene Expression Common to Acquired and Intrinsic Resistance to BRAF Inhibitor Revealed by RNA-Seq Analysis.

Melanoma cells have been shown to respond to BRAF inhibitors; however, intrinsic and acquired resistance limits their clinical application. In this study, we performed RNA-Seq analysis with BRAF inhibitor-sensitive (A375P) and -resistant (A375P/Mdr with acquired resistance and SK-MEL-2 with intrinsic resistance) melanoma cell lines, to reveal the genes and pathways potentially involved in intrinsic and acquired resistance to BRAF inhibitors. A total of 546 differentially expressed genes (DEGs), including 239 up-regulated and 307 down-regulated genes, were identified in both intrinsic and acquired resistant cells. Gene ontology (GO) analysis revealed that the top 10 biological processes associated with these genes included angiogenesis, immune response, cell adhesion, antigen processing and presentation, extracellular matrix organization, osteoblast differentiation, collagen catabolic process, viral entry into host cell, cell migration, and positive regulation of protein kinase B signaling. In addition, using the PANTHER GO classification system, we showed that the highest enriched GOs targeted by the 546 DEGs were responses to cellular processes (ontology: biological process), binding (ontology: molecular function), and cell subcellular localization (ontology: cellular component). Ingenuity pathway analysis (IPA) network analysis showed a network that was common to two BRAF inhibitor-resistant cells. Taken together, the present study may provide a useful platform to further reveal biological processes associated with BRAF inhibitor resistance, and present areas for therapeutic tool development to overcome BRAF inhibitor resistance.

A liver-specific gene expression panel predicts the differentiation status of in vitro hepatocyte models.

Alternative cell sources, such as three-dimensional organoids and induced pluripotent stem cell-derived cells, might provide a potentially effective approach for both drug development applications and clinical transplantation. For example, the development of cell sources for liver cell-based therapy has been increasingly needed, and liver transplantation is performed for the treatment for patients with severe end-stage liver disease. Differentiated liver cells and three-dimensional organoids are expected to provide new cell sources for tissue models and revolutionary clinical therapies. However, conventional experimental methods confirming the expression levels of liver-specific lineage markers cannot provide complete information regarding the differentiation status or degree of similarity between liver and differentiated cell sources. Therefore, in this study, to overcome several issues associated with the assessment of differentiated liver cells and organoids, we developed a liver-specific gene expression panel (LiGEP) algorithm that presents the degree of liver similarity as a "percentage." We demonstrated that the percentage calculated using the LiGEP algorithm was correlated with the developmental stages of in vivo liver tissues in mice, suggesting that LiGEP can correctly predict developmental stages. Moreover, three-dimensional cultured HepaRG cells and human pluripotent stem cell-derived hepatocyte-like cells showed liver similarity scores of 59.14% and 32%, respectively, although general liver-specific markers were detected. CONCLUSION: Our study describes a quantitative and predictive model for differentiated samples, particularly liver-specific cells or organoids; and this model can be further expanded to various tissue-specific organoids; our LiGEP can provide useful information and insights regarding the differentiation status of in vitro liver models. (Hepatology 2017;66:1662-1674).

Upregulation of MicroRNA-1246 Is Associated with BRAF Inhibitor Resistance in Melanoma Cells with Mutant BRAF.

PURPOSE: Intrinsic and acquired resistance limit the therapeutic benefits of inhibitors of oncogenic BRAF in melanoma. To identify microRNAs (miRNAs) associated with resistance to a BRAF inhibitor, we compared miRNA expression levels in three cell lines with different BRAF inhibitor sensitivity. MATERIALS AND METHODS: miRNA microarray analysis was conducted to compare miRNA expression levels. Real-time quantitative reverse-transcription polymerase chain reaction (qRT-PCR) was performed to confirm the expression of differentially expressed miRNAs. The cellular effects of miR-1246 were further examined by MTT assay, immunoblotting analysis, cell cycle analysis, flow cytometric assay of apoptosis, and autophagy assay. RESULTS: The miRNA microarray analysis and qRT-PCR identified five miRNAs (miR-3617, miR-92a-1, miR-1246, miR-193b-3p, and miR-17-3p) with expression that was consistently altered in two BRAF inhibitor-resistant cell lines. Among the five miRNAs, a miR-1246 mimic significantly reduced the antiproliferative effects of the BRAF inhibitor PLX4720 in BRAF inhibitor-resistant A375P (A375P/Mdr) cells, suggesting that miR-1246 upregulation confers acquired resistance to BRAF inhibition. In particular, apoptosis was identified as a major type of cell death in miR-1246-transfected cells; however, necrosis predominated in mimic-control-transfected cells, indicating that the resistance to PLX4720 in miR-1246 mimic-transfected cells is predominantly due to a reduction in necrosis. Furthermore, we found that miR-1246 promoted G2/M arrest through autophagy as a way to escape cell death by necrosis and apoptosis in response to PLX4720. The promotion of BRAF inhibitor resistance by miR-1246 was associated with lowered levels of p-ERK. CONCLUSION: These results suggest that miR-1246 may be a potential therapeutic target in melanoma with acquired resistance to BRAF inhibitors.

Novel indazole-based small compounds enhance TRAIL-induced apoptosis by inhibiting the MKK7-TIPRL interaction in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the most malignant tumors. Although various treatments, such as surgery and chemotherapy, have been developed, a novel alternative therapeutic approach for HCC therapy is urgently needed. Tumor necrosis factor-related apoptosis inducing ligand (TRAIL) is a promising anti-cancer agent, but many cancer cells are resistant to TRAIL-induced apoptosis. To help overcome TRAIL resistance in HCC cancer cells, we have identified novel chemical compounds that act as TRAIL sensitizers. We first identified the hit compound, TRT-0002, from a chemical library of 6,000 compounds using a previously developed high-throughput enzyme-linked immunosorbent assay (ELISA) screening system, which was based on the interaction of mitogen-activated protein kinase kinase 7 (MKK7) and TOR signaling pathway regulator-like (TIPRL) proteins and a cell viability assay. To increase the efficacy of this TRAIL sensitizer, we synthesized 280 analogs of TRT-0002 and finally identified two lead compounds (TRT-0029 and TRT-0173). Co-treating cultured Huh7 cells with either TRT-0029 or TRT-0173 and TRAIL resulted in TRAIL-induced apoptosis due to the inhibition of the MKK7-TIPRL interaction and subsequent phosphorylation of MKK7 and c-Jun N-terminal kinase (JNK). In vivo, injection of these compounds and TRAIL into HCC xenograft tumors resulted in tumor regression. Taken together, our results suggest that the identified lead compounds serve as TRAIL sensitizers and represent a novel strategy to overcome TRAIL resistance in HCC.

Plasma glutamate carboxypeptidase is a negative regulator in liver cancer metastasis.

Tumor metastasis is the leading cause of cancer death. In the metastatic process, EMT is a unique phenotypic change that plays an important role in cell invasion and changes in cell morphology. Despite the clinical significance, the mechanism underlying tumor metastasis is still poorly understood. Here we report a novel mechanism by which secreted plasma glutamate carboxypeptidase(PGCP) negatively involves Wnt/ß-catenin signaling by DKK4 regulation in liver cancer metastasis. Pathway analysis of the RNA sequencing data showed that PGCP knockdown in liver cancer cell lines enriched the functions of cell migration, motility and mesenchymal cell differentiation. Depletion of PGCP promoted cell migration and invasion via activation of Wnt/ß-catenin signaling pathway components such as phospho-LRP6 and ß-catenin. Also, addition of DKK4 antagonized the Wnt/ß-catenin signaling cascade in a thyroxine (T4)-dependent manner. In an in vivo study, metastatic nodules were observed in the lungs of the mice after injection of shPGCP stable cell lines. Our findings suggest that PGCP negatively associates with Wnt/ß-catenin signaling during metastasis. Targeting this regulation may represent a novel and effective therapeutic option for liver cancer by preventing metastatic activity of primary tumor cells.

Induction of Resistance to BRAF Inhibitor Is Associated with the Inability of Spry2 to Inhibit BRAF-V600E Activity in BRAF Mutant Cells.

The clinical benefits of oncogenic BRAF inhibitor therapies are limited by the emergence of drug resistance. In this study, we investigated the role of a negative regulator of the MAPK pathway, Spry2, in acquired resistance using BRAF inhibitor-resistant derivatives of the BRAF-V600E melanoma (A375P/Mdr). Real-time RT-PCR analysis indicated that the expression of Spry2 was higher in A375P cells harboring the BRAF V600E mutation compared with wild-type BRAF-bearing cells (SK-MEL-2) that are resistant to BRAF inhibitors. This result suggests the ability of BRAF V600E to evade feedback suppression in cell lines with BRAF V600E mutations despite high Spry2 expression. Most interestingly, Spry2 exhibited strongly reduced expression in A375P/Mdr cells with acquired resistance to BRAF inhibitors. Furthermore, the overexpression of Spry2 partially restored sensitivity to the BRAF inhibitor PLX4720 in two BRAF inhibitor-resistant cells, indicating a positive role for Spry2 in the growth inhibition induced by BRAF inhibitors. On the other hand, long-term treatment with PLX4720 induced pERK reactivation following BRAF inhibition in A375P cells, indicating that negative feedback including Spry2 may be bypassed in BRAF mutant melanoma cells. In addition, the siRNA-mediated knockdown of Raf-1 attenuated the rebound activation of ERK stimulated by PLX4720 in A375P cells, strongly suggesting the positive role of Raf-1 kinase in ERK activation in response to BRAF inhibition. Taken together, these data suggest that RAF signaling may be released from negative feedback inhibition through interacting with Spry2, leading to ERK rebound and, consequently, the induction of acquired resistance to BRAF inhibitors.

Oncogenic BRAF inhibitor UAI-201 induces cell cycle arrest and autophagy in BRAF mutant glioma cells.

AIMS: An activating mutation of BRAF (BRAF-V600E) has been reported in a subset of malignant brain tumors. Thus, the aim of the present study was to identify the antiproliferative effect of the new oncogenic B-Raf targeting drug UAI-201 on 6 types of glioma cell lines with differing B-Raf mutational status. MAIN METHODS: The IC50 values of UAI-201 were determined using crystal violet assays in six glioma cell lines. Real-time RT-PCR was performed to assess the functional role of multidrug resistance proteins in response to UAI-201. The effects of UAI-201 on six glioma cells were further examined by immunoblotting analysis, cell cycle analysis, flow cytometric apoptotic assay and autophagy assay. To identify the role of autophagy in UAI-201-induced growth inhibition, Atg5 and Beclin 1 were knocked down by RNA interference. KEY FINDINGS: Real-time RT-PCR analysis showed a poor correlation between UAI-201 activity and the expression level of multidrug resistance proteins. The growth inhibitory effects of UAI-201 correlated with the BRAF-V600E genotype of the glioma cell lines. BRAF blockade with UAI-201 resulted in dose-dependent inhibition of MEK/ERK phosphorylations and increased G0/G1 arrest in glioma cells with BRAF-V600E. Interestingly, UAI-201 preferentially induced autophagy in BRAF-V600E cells, but not in BRAF-WT cells. More notably, autophagy inhibition through siRNA-mediated Beclin 1 knockdown partially attenuated the growth inhibition induced by UAI-201 in BRAF-V600E cells. SIGNIFICANCE: The pro-death autophagic processes could be one of the underlying mechanisms for the sensitization of BRAF-V600E glioma cells toward UAI-201.

The siRNA-mediated downregulation of N-Ras sensitizes human melanoma cells to apoptosis induced by selective BRAF inhibitors.

The clinical benefit of selective BRAF inhibitor therapies is limited by the emergence of drug resistance. Here, we investigated the molecular basis underlying the acquired resistance to a BRAF inhibitor by comparing the signaling pathways in the parental A375P cells and the resistant subline (A375P/Mdr). We demonstrate that MAPK re-activation does not contribute to the mechanism of resistance to UAI-201 of A375P/Mdr cells. The relative quantitative analysis using the 2(-ΔΔCt) method revealed that the BRAF inhibitor resistance observed in A375P/Mdr cells is not mediated through the overexpression of MDR proteins. In particular, we found that the expression of N-Ras was upregulated in BRAF inhibitor-resistant A375P/Mdr cells compared with A375P cells. In fact, siRNA-mediated N-Ras knockdown partially conferred UAI-201 sensitivity to A375P/Mdr cells, implying that N-Ras upregulation confers acquired resistance to BRAF inhibition. Notably, the flow cytometric analysis of the N-Ras-knockdown A375P/Mdr cells revealed that UAI-201 causes a significant accumulation of cells in the G 0/G 1 phase with a concomitant decrease in the number of cells in the S and G 2/M phases. However, platelet-derived growth factor receptor ß (PDGFRß) knockdown failed to sensitize A375P/Mdr cells to growth suppression by UAI-201, although a remarkable increase in PDGFRß was observed in the A375P cells after UAI-201 treatment. Taken together, our results suggest that N-Ras is worth targeting to improve the therapeutic outcome of melanomas with acquired resistance to BRAF inhibitors.

Autophagy-Dependent Survival of Mutant B-Raf Melanoma Cells Selected for Resistance to Apoptosis Induced by Inhibitors against Oncogenic B-Raf.

Most patients with mutant B-Raf melanomas respond to inhibitors of oncogenic B-Raf but resistance eventually emerges. To better understand the mechanisms that determine the long-term responses of mutant B-Raf melanoma cells to B-Raf inhibitor, we used chronic selection to establish B-Raf (V600E) melanoma clones with acquired resistance to the new oncogenic B-Raf inhibitor UI-152. Whereas the parental A375P cells were highly sensitive to UI-152 (IC50<0.5 µM), the resistant sub-line (A375P/Mdr) displayed strong resistance to UI-152 (IC50>20 µM). Immunofluorescence analysis indicated the absence of an increase in the levels of P-glycoprotein multidrug resistance (MDR) transporter in A375P/Mdr cells, suggesting that resistance was not attributable to P-glycoprotein overexpression. In UI-152-sensitive A375P cells, the anti-proliferative activity of UI-152 appeared to be due to cell-cycle arrest at G0/G1 with the induction of apoptosis. However, we found that A375P/Mdr cells were resistant to the apoptosis induced by UI-152. Interestingly, UI-152 preferentially induced autophagy in A375P/Mdr cells but not in A375P cells, as determined by GFP-LC3 puncta/cell counts. Further, autophagy inhibition with 3-methyladenine (3-MA) partially augmented growth inhibition of A375P/Mdr cells by UI-152, which implies that a high level of autophagy may protect UI-152-treated cells from undergoing growth inhibition. Together, our data implicate high rates of autophagy as a key mechanism of acquired resistance to the oncogenic B-Raf inhibitor, in support of clinical studies in which combination therapy with autophagy targeted drugs is being designed to overcome resistance.

Defective autophagy in multidrug resistant cells may lead to growth inhibition by BH3-mimetic gossypol.

The clinical efficacy of many chemotherapeutic agents has been reduced due to the development of drug resistance. In this article, we aimed to validate gossypol, a natural BH3 mimetic found in cottonseeds, as a potential therapeutic to overcome multidrug resistance (MDR). Gossypol was found to retain its efficacy in v-Ha-ras-transformed NIH 3T3 cells that overexpressed P-glycoprotein (Ras-NIH 3T3/Mdr), which was similar to the efficacy observed in their parental counterparts (Ras-NIH 3T3). A rhodamine assay revealed that the alteration of MDR activity did not contribute to the cytotoxic effect of gossypol. Gossypol caused a G2 /M arrest by the induction of p21(Cip1) and the down-regulation of p27(Kip1) expression in Ras-NIH 3T3 cells, whereas no significant G2 /M arrest was exhibited in Ras-NIH 3T3/Mdr cells. Surprisingly, a 48-h treatment with gossypol induced apoptotic cell death in Ras-NIH 3T3 cells; however, gossypol induced both apoptosis and necrosis in Ras-NIH 3T3/Mdr cells, as determined with flow cytometry analysis. More notably, gossypol preferentially induced autophagy in Ras-NIH 3T3 cells but not in Ras-NIH 3T3/Mdr cells. Coimmunoprecipitation and flow cytometric analysis revealed that gossypol-induced autophagy is independent of the dissociation of Beclin 1 from Bcl-2 in Ras-NIH 3T3 cells. Taken together, these results suggest that the antiproliferative activity of gossypol appears to be due to cell-cycle arrest at the G2 /M phase, with the induction of apoptosis in Ras-NIH 3T3 cells. In addition, defective autophagy might contribute to apoptotic and necrotic cell death in response to gossypol in Ras-NIH 3T3/Mdr cells.

The role of autophagy in cytotoxicity induced by new oncogenic B-Raf inhibitor UI-152 in v-Ha-ras transformed fibroblasts.

In human cancers, B-Raf is the most frequently mutated protein kinase in the MAPK signaling cascade, making it an important therapeutic target. We recently discovered a potent and selective B-Raf inhibitor, UI-152, by using a structure-based drug design strategy. In this study, we examined whether B-Raf inhibition by UI-152 may be an effective therapeutic strategy for eliminating cancer cells transformed with v-Ha-ras (Ras-NIH 3T3). UI-152 displayed selective cytotoxicity toward Ras-NIH 3T3 cells while having little to no effect on non-transformed NIH 3T3 cells. We found that treatment with UI-152 markedly increased autophagy and, to a lesser extent, apoptosis. However, inhibition of autophagy by addition of 3-MA failed to reverse the cytotoxic effects of UI-152 on Ras-NIH 3T3 cells, demonstrating that apoptosis and autophagy can act as cooperative partners to induce growth inhibition in Ras-NIH 3T3 cells treated with UI-152. Most interestingly, cell responses to UI-152 appear to be paradoxical. Here, we showed that although UI-152 inhibited ERK, it induced B-Raf binding to Raf-1 as well as Raf-1 activation. This paradoxical activation of Raf-1 by UI-152 is likely to be coupled with the inhibition of the mTOR pathway, an intracellular signaling pathway involved in autophagy. We also showed for the first time that, in multi-drug resistant cells, the combination of UI-152 with verapamil significantly decreased cell proliferation and increased autophagy. Thus, our findings suggest that the inhibition of autophagy, in combination with UI-152, offers a more effective therapeutic strategy for v-Ha-ras-transformed cells harboring wild-type B-Raf.