BackgroundCancer stem cells (CSCs) are a small subpopulation of tumor cells with the capability of self-renewal and drug resistance, leading to tumor progression and disease relapse. Our study aimed to investigate the antitumor effect of berbamine, extracted from berberis amurensis, on prostate CSCs.MethodsSphere formation was used to collect prostate CSCs. The viability, proliferation, invasion, migration, and apoptosis assays were used to evaluate the antitumor effect of berbamine on prostate CSCs. Prostate CSC markers were analyzed by flow cytometry and qRT-PCR. Small RNA sequencing analysis was conducted to analyse miRNAs. Exosomes were extracted using the ExoQuick-TC kit and verified by testing exosomal markers using western blot.ResultsBerbamine targets prostate CSCs. Additionally, berbamine enhanced the antitumor effect of cabazitaxel, a second-line chemotherapeutic drug for advanced prostate cancer, and re-sensitized Cabazitaxel-resistant PCa cells (CabaR-DU145) to cabazitaxel by inhibiting ABCG2, CXCR4, IGF2BP1, and p-STAT3. Berbamine enhanced the expression of let-7 miRNA family and miR-26b and influenced the downstream targets IGF2BP1 and p-STAT3, respectively. Silencing CXCR4 and ABCG2 downregulated the expression of IGF2BP1 and p-STAT3, respectively. Importantly, berbamine enhanced also levels of exosomal let-7 family and miR-26b, suggesting that berbamine possibly influences the expression of let-7 family and miR-26b through exosome delivery. Exosomes derived from berbamine-treated CabaR-DU145 cells re-sensitized the cells to cabazitaxel.ConclusionBerbamine enhanced the toxic activity of cabazitaxel and reversed cabazitaxel resistance potentially through CXCR4/exosomal let-7/IGF2BP1 and ABCG2/exosomal miR-26b/p-STAT3 axes.

Background Management of clear cell renal cell carcinoma (ccRCC) has changed rapidly in recent years with the advent of immune checkpoint inhibitors (ICIs). However, only a limited number of patients can sustainably respond to immune checkpoint inhibitors and many patients develop resistance to therapy, creating an additional need for therapeutic strategies to improve the efficacy of systemic therapies. Methods Binding probability and target genes prediction using online databases, invasion, migration, and apoptosis assays as well as the inhibition of cancer stem cells (CSCs) markers in ccRCC cell lines were used to select the most promising phytochemicals (PTCs). Mixed lymphocyte tumor cell culture (MLTC) system and flow cytometry were performed to confirm the potential combination strategy. The potential immunotherapeutic targets and novel CSC markers were identified via the NanoString analysis. The mRNA and protein expression, immune signatures as well as survival characteristics of the marker in ccRCC were analyzed via bioinformation analysis. Results Shikonin was selected as the most promising beneficial combination partner among 11 PTCs for ipilimumab for the treatment of ccRCC patients due to its strong inhibitory effect on CSCs, the significant reduction of FoxP3 + Treg cells in peripheral blood mononuclear cells (PBMCs) of patients and activation of the endogenous effector CD3 + CD8 + and CD3 + CD4 + T cells in response to the recognition of tumor specific antigens. Based on NanoString analysis VCAM1, CXCL1 and IL8 were explored as potential immunotherapeutic targets and novel CSC markers in ccRCC. The expression of VCAM1 was higher in the tumor tissue both at mRNA and protein levels in ccRCC compared with normal tissue, and was significantly positively correlated with immune signatures and survival characteristics in ccRCC patients. Conclusion We propose that a combination of shikonin and ipilimumab could be a promising treatment strategy and VCAM1 a novel immunotherapeutic target for the treatment of ccRCC.

Glioblastoma multiforme is a malignant neoplasia with a median survival of less than two years and without satisfactory therapeutic options. The so-called glioblastoma stem cells escape the established radio- and chemotherapies and lead to tumor recurrence in most cases. The alkaloid Shikonin with its various anti stem cell properties and the interstitial photodynamic therapy with 5-aminolevulinic acid seem to be promising new options in the therapy of glioblastoma. In this study, in vitro investigations were performed to observe the influence of Shikonin on viability, proliferation, induction of apoptosis and the capability of forming tumor spheres in U-87 MG and the primary glioblastoma cell line GB14. The combined effect with the chemotherapeutic temozolomide and photodynamic treatment on the mRNA expression of glioma specific stem cell markers and further examined intracellular protoporphyrin IX accumulation under Shikonin treatment was analyzed. Shikonin effectively inhibited the capability of forming tumor spheres and enhanced temozolomide effectiveness in the reduction of proliferation and in the induction of apoptosis. Additionally, Shikonin increased the mRNA expression of the tumor suppressing Neurofibromatosis type 1 (NF1) gene and showed modulating effects on intracellular protoporphyrin IX.

Emerging evidence from research or clinical studies reported that ABCG2 (ATP-binding cassette sub-family G member 2) interrelates with multidrug resistance (MDR) development in cancers. However, no comprehensive pan-cancer analysis is available at present. Therefore, we explore multiple databases, such as TCGA to investigate the potential therapeutic roles of ABCG2 across 33 different tumors. ABCG2 is expressed on a lower level in most cancers and shows a protective effect. For example, a lower expression level of ABCG2 was detrimental to the survival of adrenocortical carcinoma (TCGA-ACC), glioblastoma multiforme (GBM), and kidney renal clear cell carcinoma (KIRC) patients. Distinct associations exist between ABCG2 expression and stemness scores, microenvironmental scores, microsatellite instability (MSI), and tumor mutational burden (TMB) of tumor patients. We observed a significant positive correlation between the ABCG2 mutation site and prognosis in uterine corpus endometrial carcinoma (UCEC) patients. Moreover, transmembrane transporter activity and hormone biosynthetic-associated functions were found to be involved in the functionality of ABCG2 and its related genes. The cDNAs of cancer cell lines were collected to detect exon mutation sequences and to analyze ABCG2 mRNA expression. The mRNA expression level of ABCG2 showed a significant difference among spheres and drug-resistant cancer cell lines compared with their corresponding adherent cancer cell lines in six types of cancer. This pan-cancer study provides, for the first time, a comprehensive understanding of the multifunctionality of ABCG2 and unveils further details of the potential therapeutic role of ABCG2 in pan-cancer.

The aim of the study was to develop a new therapeutic strategy to target cancer stem cells (CSCs) in clear cell renal cell carcinoma (ccRCC) and to identify typical CSC markers to improve therapy effectiveness. It was found that the corrected-mRNA expression-based stemness index was upregulated in kidney renal clear cell carcinoma (KIRC) tissues compared to non-tumor tissue and increased with higher tumor stage and grade. EZH2 was identified as a CSC marker and prognosis factor for KIRC patients. The expression of EZH2 was associated with several activated tumor-infiltrating immune cells. High expression of EZH2 was enriched in immune-related pathways, low expression was related to several metabolic pathways. Epigallocatechin-3-gallate (EGCG) was identified as the most potent suppressor of EZH2, was able to inhibit viability, migration, and invasion, and to increase the apoptosis rate of ccRCC CSCs. KIF11, VEGF, and MMP2 were identified as predictive EGCG target genes, suggesting a potential mechanism of how EZH2 might regulate invasiveness and migration. The percentages of FoxP3+ Treg cells in the peripheral blood mononuclear cells of ccRCC patients decreased significantly when cultured with spheres pretreated with EGCG plus sunitinib compared to spheres without treatment. Our findings provide new insights into the treatment options of ccRCC based on targeting CSCs.

Cancer stem cells (CSCs) are a small population among cancer cells, defined as capable of self-renewal, and driving tumor growth, metastasis, and therapeutic relapse. The development of therapeutic strategies to target CSCs is of great importance to prevent tumor metastasis and relapse. Increasing evidence shows that shikonin has inhibiting effects on CSCs. This study was to determine the effect of shikonin on prostate CSCs, and on drug resistant cells. Sphere formation assay was used to enrich prostate CSCs. The effect of shikonin on viability, proliferation, migration, and invasion was studied. Typical CSCs markers were analyzed by flow cytometry and RT-qPCR. The cytotoxic mechanism of shikonin was analyzed by staining for annexin V, reactive oxygen species (ROS) and mitochondrial membrane potential. To study the effect of shikonin on drug-resistant cells a cabazitaxel resistant cell line was established. Shikonin inhibited the viability, proliferation, migration, and invasion of prostate CSCs. Shikonin enhanced the antitumor effect of cabazitaxel, which is a second-line chemotherapeutic drug in advanced prostate cancer. Shikonin induced apoptosis through generating ROS and disrupting the mitochondrial membrane potential. Furthermore, shikonin suppressed the expression of ALDH3A1 and ABCG2 in prostate CSCs, which are two markers related to drug-resistance. When inhibiting the expression of ABCG2 and ALDH3A1, the cabazitaxel resistant cells acquired more sensibility to cabazitaxel. Shikonin enhances the cytotoxic activity of cabazitaxel in prostate CSCs and reverses the cabazitaxel-resistant state.

You have accessJournal of UrologyKidney Cancer: Basic Research & Pathophysiology II1 Apr 2018MP72-12 CO-TREATMENT WITH L-METHADONE INCREASES THE EFFICACY OF ANTITUMOR AGENTS IN RENAL CELL CARCINOMA CELLS IN VITRO Birgit Stadlbauer, Heike Pohla, Christian Stief, and Alexander Buchner Birgit StadlbauerBirgit Stadlbauer More articles by this author , Heike PohlaHeike Pohla More articles by this author , Christian StiefChristian Stief More articles by this author , and Alexander BuchnerAlexander Buchner More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2018.02.2296AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES In leukemia and glioblastoma cells, a significant increase of apoptosis rates was observed in vitro under cytostatic therapy by activation of the μ-opioid receptor with methadone. This study investigated the effect of methadone co-treatment in renal cell carcinoma (RCC) cells in the presence of different antitumor agents. METHODS The clear-cell RCC cell line RCC-26 was incubated with different antitumor drugs (doxorubicin, mitoxantrone, axitinib and others). The ratio of cells in apoptosis after 5 days was analyzed using flow cytometry with annexin V-APC and 7AAD double staining. In each setting, the cells were co-incubated with various concentrations of L-methadone and D,L-methadone (0-30μg/ml). The opioid antagonist naloxone served as negative control. Additionally, cell proliferation assays (Cell Titer 96 AQueous One Solution, Promega) and cell migration assays (Automated Cellular Analysis System, MetaVi Labs) were conducted. Furthermore, gene expression analysis including pathway analysis was performed using microarrays (Affymetrix GeneChip Prime View with ca. 49,000 transcripts) to reveal changes in cell biology caused by methadone co-treatment. RESULTS Incubation of RCC-26 cells with 1μM doxorubicin resulted in 32% apoptosis after 5 days. Co-incubation with L-methadone showed a dose-dependent increase of apoptosis rate up to 78% (2.4-fold increase). The effect of D,L-methadone was similar but less pronounced. Control experiments with naloxone instead of methadone showed no influence on the apoptosis rate. Comparable results were achieved with other tumor cell lines, e.g. A172 glioblastoma cells. RCC-26 proliferation was significantly decreased by co-incubation with L-methadone and doxorubicin vs. one of these drugs alone. Cell migration assays showed a complete inhibition of RCC-26 migration by co-incubation with 10ng/ml L-methadone and 1μM doxorubicin. Microarray analysis identified 333 upregulated and 69 downregulated genes (≥2-fold change of expression level, p<0.05) caused by L-methadone in RCC-26 cells. CONCLUSIONS Co-incubation with L-methadone enhances the therapeutic effect of antitumor drugs in renal cell carcinoma cells and in various other tumor cell lines. The increase of the cytostatic effect depends on the combination of cell line and antitumor agent. The combination of L-methadone with certain antitumor drugs might be a promising new approach to increase the therapeutic efficacy in renal cell carcinoma. © 2018FiguresReferencesRelatedDetails Volume 199Issue 4SApril 2018Page: e956 Advertisement Copyright & Permissions© 2018MetricsAuthor Information Birgit Stadlbauer More articles by this author Heike Pohla More articles by this author Christian Stief More articles by this author Alexander Buchner More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

You have accessJournal of UrologyProstate Cancer: Basic Research & Pathophysiology II1 Apr 2017MP83-19 CO-TREATMENT WITH L-METHADONE SIGNIFICANTLY INCREASES THE EFFICACY OF CYTOSTATIC DRUGS IN PROSTATE CANCER CELLS Birgit Stadlbauer, Detlef Kozian, Christian Stief, and Alexander Buchner Birgit StadlbauerBirgit Stadlbauer More articles by this author , Detlef KozianDetlef Kozian More articles by this author , Christian StiefChristian Stief More articles by this author , and Alexander BuchnerAlexander Buchner More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2017.02.2587AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES In leukemia and glioblastoma cells, a significant increase of apoptosis rates has been observed in vitro under cytostatic therapy by activation of the µ-opioid receptor with methadone. This study investigated the effect of opioid receptor activation in prostate cancer cells in presence of different cytotoxic drugs. METHODS The prostate cancer cell lines PC-3, DU145 and LNCaP as well as control cell lines RCC-26 and A172 (renal cell carcinoma and glioblastoma) were incubated with different concentrations of the cytostatic drugs cabazitaxel and doxorubicin and the number of cells in apoptosis after 3 and 5 days was analyzed using flow cytometry (with annexin V-APC and 7AAD staining). In addition, the cells were incubated with various concentrations of L-methadone (0-100 µg/ml). Furthermore, gene expression analysis was performed using microarrays (Affymetrix GeneChip Prime View, ca. 49,000 transcripts) to detect changes in cell biology caused by methadone treatment. RESULTS Incubation of PC-3 cells with 10 nM cabazitaxel resulted in 53% apoptosis after 5 days. This rate remained constant under co-incubation with L-methadone. Incubation with 0.3 μM doxorubicin resulted in 37% apoptosis after 5 days. In this setting, co-incubation with L-methadone showed a dose-dependent increase of apoptosis rate up to 88% (2.4-fold increase). Control experiments with fentanyl and naloxone instead of methadone showed no influence on apoptosis rate. Comparable and partly even clearer results were achieved with DU145, LNCaP, RCC-26 and A172 cells: the cabazitaxel-induced apoptosis rates remained stable under L-methadone, the doxorubicin-induced apoptosis was significantly increased by L-methadone (up to 3.8-fold increase). Microarray analysis revealed 128 upregulated and 398 downregulated genes (≥2-fold change of expression level, p<0.05) caused by L-methadone in PC-3 cells. CONCLUSIONS Stimulation of the µ-opioid receptors by L-methadone enhances the therapeutic effect of cytostatic drugs in prostate carcinoma cells and in other tumor cell lines. The increase of cytostatic effect depends on the combination of cell line and cytostatic agent. The combination of L-methadone with certain cytotoxic drugs can be a promising new approach to increase the therapeutic efficacy in hormone-refractory prostate carcinoma. © 2017FiguresReferencesRelatedDetails Volume 197Issue 4SApril 2017Page: e1114 Advertisement Copyright & Permissions© 2017MetricsAuthor Information Birgit Stadlbauer More articles by this author Detlef Kozian More articles by this author Christian Stief More articles by this author Alexander Buchner More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Our previously reported phase I clinical trial with the allogeneic gene-modified tumor cell line RCC-26/CD80/IL-2 showed that vaccination was well tolerated and feasible in metastatic renal cell carcinoma (RCC) patients. Substantial disease stabilization was observed in most patients despite a high tumor burden at study entry. To investigate alterations in immune responses that might contribute to this effect, we performed an extended immune monitoring that included analysis of reactivity against multiple antigens, cytokine/chemokine changes in serum and determination of the frequencies of immune suppressor cell populations, including natural regulatory T cells (nTregs) and myeloid-derived suppressor cell subsets (MDSCs). An overall immune response capacity to virus-derived control peptides was present in 100% of patients before vaccination. Vaccine-induced immune responses to tumor-associated antigens occurred in 75% of patients, demonstrating the potent immune stimulatory capacity of this generic vaccine. Furthermore, some patients reacted to peptide epitopes of antigens not expressed by the vaccine, showing that epitope-spreading occurred in vivo. Frequencies of nTregs and MDSCs were comparable to healthy donors at the beginning of study. A significant decrease of nTregs was detected after vaccination (p = 0.012). High immune response rates, decreased frequencies of nTregs and a mixed T helper 1/T helper 2 (TH1/TH2)-like cytokine pattern support the applicability of this RCC generic vaccine for use in combination therapies.

Background T-cell mediated immunity likely plays an important role in controlling HIV-1 infection and progression to AIDS. Several candidate vaccines against HIV-1 aim at stimulating cellular immune responses, either alone or together with the induction of neutralizing antibodies, and assays able to measure CD8 and CD4 T-cell responses need to be implemented. At present, the IFN-γ-based ELISPOT assay is considered the gold standard and it is broadly preferred as primary assay for detection of antigen-specific T-cell responses in vaccine trials. However, in spite of its high sensitivity, the measurement of the sole IFN-γ production provides limited information on the quality of the immune response. On the other hand, the introduction of polychromatic flow-cytometry-based assays such as the intracellular cytokine staining (ICS) strongly improved the capacity to detect several markers on a single cell level. Results The cumulative analysis of 275 samples from 31 different HIV-1 infected individuals using an ICS staining procedure optimized by our laboratories revealed that, following antigenic stimulation, IFN-γ producing T-cells were also producing MIP-1β whereas T-cells characterized by the sole production of IFN-γ were rare. Since the analysis of the combination of two functions decreases the background and the measurement of the IFN-γ+ MIP-1β+ T-cells was equivalent to the measurement of the total IFN-γ+ T-cells, we adopted the IFN-γ+ MIP-1β+ data analysis system to evaluate IFN-γ-based, antigen-specific T-cell responses. Comparison of our ICS assay with ELISPOT assays performed in two different experienced laboratories demonstrated that the IFN-γ+ MIP-1β+ data analysis system increased the sensitivity of the ICS up to levels comparable to the sensitivity of the ELISPOT assay. Conclusion The IFN-γ+ MIP-1β+ data evaluation system provides a clear advantage for the detection of low magnitude HIV-1-specific responses. These results are important to guide the choice for suitable highly sensitive immune assays and to build reagent panels able to accurately characterize the phenotype and function of responding T-cells. More importantly, the ICS assay can be used as primary assay to evaluate HIV-1-specific responses without losing sensitivity in comparison to the ELISPOT assay.