Decrease Drug Resistance Research Articles

Detecting Mutations in the Mycobacterium tuberculosis Pyrazinamidase Gene pncA to Improve Infection Control and Decrease Drug Resistance Rates in Human Immunodeficiency Virus Coinfection.

Hospital infection control measures are crucial to tuberculosis (TB) control strategies within settings caring for human immunodeficiency virus (HIV)–positive patients, as these patients are at heightened risk of developing TB. Pyrazinamide (PZA) is a potent drug that effectively sterilizes persistent Mycobacterium tuberculosis bacilli. However, PZA resistance associated with mutations in the nicotinamidase/pyrazinamidase coding gene, pncA, is increasing. A total of 794 patient isolates obtained from four sites in Lima, Peru, underwent spoligotyping and drug resistance testing. In one of these sites, the HIV unit of Hospital Dos de Mayo (HDM), an isolation ward for HIV/TB coinfected patients opened during the study as an infection control intervention: circulating genotypes and drug resistance pre- and postintervention were compared. All other sites cared for HIV-negative outpatients: genotypes and drug resistance rates from these sites were compared with those from HDM. HDM patients showed high concordance between multidrug resistance, PZA resistance according to the Wayne method, the two most common genotypes (spoligotype international type [SIT] 42 of the Latino American-Mediterranean (LAM)-9 clade and SIT 53 of the T1 clade), and the two most common pncA mutations (G145A and A403C). These associations were absent among community isolates. The infection control intervention was associated with 58–92% reductions in TB caused by SIT 42 or SIT 53 genotypes (odds ratio [OR] = 0.420, P = 0.003); multidrug-resistant TB (OR = 0.349, P < 0.001); and PZA-resistant TB (OR = 0.076, P < 0.001). In conclusion, pncA mutation typing, with resistance testing and spoligotyping, was useful in identifying a nosocomial TB outbreak and demonstrating its resolution after implementation of infection control measures.

Competitive binding of (−)-epigallocatechin-3-gallate and 5-fluorouracil to human serum albumin: A fluorescence and circular dichroism study

Combination therapy with more than one therapeutic agent can improve therapeutic efficiency and decrease drug resistance. In this study, the interactions of human serum albumin (HSA) with individual or combined anticancer drugs, (−)-epigallocatechin-3-gallate (EGCG) and 5-fluorouracil (FU), were investigated by fluorescence and circular dichroism (CD) spectroscopy. The results demonstrated that the interaction of EGCG or FU with HSA is a process of static quenching and EGCG formed a more stable complex. The competitive experiments of site markers suggested that both anti-carcinogens mainly bound to site I (subdomain IIA). The interaction forces which play important roles in the binding process were discussed based on enthalpy and entropy changes. Moreover, the competition binding model for a ternary system was proposed so as to precisely calculate the binding parameters. The results demonstrated that one drug decreased the binding affinity of another drug with HSA, resulting in the increasing free drug concentration at the action sites. CD studies indicated that there was an alteration in HSA secondary structure due to the binding of EGCG and FU. It can be concluded that the combination of EGCG with FU may enhance anticancer efficacy. This finding may provide a theoretical basis for clinical treatments.

NF-κB Blockers Gifted by Mother Nature: Prospectives in Cancer Cell Chemosensitization.

Despite the significant developments made in the field of diagnosis and treatment modalities of cancer during the last two decades, it still remains one of the most life threatening diseases killing 8.2 million people annually across the globe. It has been well-established that development of chemoresistance in cancer cells is the major cause of failure of chemotherapeutic agents in clinic. Most of the chemotherapeutic agents currently being used activate NF-κB and NF-κB regulated gene products in cancer cells and induce drug resistance. Increasing lines of evidences suggest that NF-κB blockers have high potential in decreasing drug resistance and sensitize cancer cells to chemotherapeutic agents. A through literature search was carried out in pubmed to identify natural NF-κB inhibitors that possess high potential in sensitizing cancer cells. Our literature search retrived a number of NF-κB inhibitors that have been identified during the last several years. Notably, the inhibitors obtained from Mother Nature such as curcumin, tocotrienol, resveratrol, garcinol etc. are found to be highly safe, efficacious and inexpensive. Many preclinical and clinical studies have revealed that these agents can block the activation of NF-κB in cancer cells to overcome drug resistance and make them sensitive to chemotherapeutic agents. Both basic and clinical research revealed that constitutive activation of NF-κB is the prime reason for inducing drug resistance in cancer cells. This comprehensive review scientifically evaluates the chemosensitizing potential of these natural agents which serve as potent NF-κB blockers, based on evidence based literature.

Targeted delivery of vitamin D3-loaded nanoparticles to C6 glioma cell line increased resistance to doxorubicin, epirubicin, and docetaxel in vitro.

In recent years, targeted delivery systems have been used along with combinatorial therapy to decrease drug resistance and increase cancer therapy efficacy. The anti-proliferative effects of vitamin D3 (VD3) on cancerous cells, such as C6 glioma, with active hedgehog pathways raised the question as to whether pre-targeting C6 glioma cells with VD3-loaded nanoparticles (VD3NPs) can enhance the anti-tumor effects of doxorubicin, epirobicin, and docetaxel on this drug-resistant cell line. Here, studying at cellular, nuclear, protein, and gene levels we demonstrated that VD3NP-doxorubicin and VD3NP-epirobicin combinations increased the probability of chemotherapy/radiotherapy resistance and cancer stem cell (CSC) properties in C6 glioma significantly (P<0.05), compared to doxorubicin and epirobicin alone. However, VD3NP-docetaxel combination may have the potential in sensitizing C6 cells to ionizing irradiation, but this combination also increased the CSC properties and the probability of drug resistance significantly (P<0.05), compared to docetaxel alone. Although our previous study showed that targeted delivery of VD3 reduced the rate of proliferation significantly (P<0.05) in C6 glioma cells (a drug-resistant cell line), here we concluded that combinatorial therapy of exogenous VD3 with doxorubicin, epirobicin, and docetaxel not only did not lead to the enhancement of cytotoxic effects of the aforementioned drugs but also increased the cancerous characteristics in C6 glioma, in vitro.

A three-drug nanoscale drug delivery system designed for preferential lymphatic uptake for the treatment of metastatic melanoma

Metastatic melanoma has a high mortality rate due to lymphatic progression of the disease. Current treatment is surgery followed by radiation and intravenous chemotherapy. However, drawbacks for current chemotherapeutics lie in the fact that they develop resistance and do not achieve therapeutic concentrations in the lymphatic system. We hypothesize that a three-drug nanoscale drug delivery system, tailored for lymphatic uptake, administered subcutaneously, will have decreased drug resistance and therefore offer better therapeutic outcomes. We prepared and characterized nanoparticles (NPs) with docetaxel, everolimus, and LY294002 in polyethyleneglycol-block-poly(ε-caprolactone) (PEG–PCL) polymer with different charge distributions by modifying the ratio of anionic and neutral end groups on the PEG block. These NPs are similarly sized (~48nm), with neutral, partially charged, or fully charged surface. The NPs are able to load ~2mg/mL of each drug and are stable for 24h. The NPs are assessed for safety and efficacy in two transgenic metastatic melanoma mouse models. All the NPs were safe in both models based on general appearance, weight changes, death, and blood biochemical analyses. The partially charged NPs are most effective in decreasing the number of melanocytes at both the proximal (sentinel) lymph node (LN) and the distal LN from the injection site. The neutral NPs are efficacious at the proximal LN, while the fully charged NPs have no effect on either LNs. Thus, our data indicates that the NP surface charge and lymphatic efficacy are closely tied to each other and the partially charged NPs have the highest potential in treating metastatic melanoma.

Quercetin Mediates β-Catenin in Pancreatic Cancer Stem-Like Cells.

The aim of this study is to explore how quercetin interacts with pancreatic cancer stem-like cells and the mechanism underlying the effective quercetin-mediated suppression. A model of pancreatic cancer stem-like cells was generated by using a sphere formation culture system. A comparative analysis was performed between the parent cells and pancreatic cancer stem-like cells with a related treatment strategy focusing on cancer stem cell (CSC) properties and drug-resistance-related mechanisms in vitro. Our data show that pancreatic cancer stem-like cells have greater resistance to gemcitabine and stronger CSC properties compared with the parent cells. In contrast to the pancreatic cancer stem-like cells, overexpression of β-catenin was observed in the parent cells. Quercetin suppressed proliferation, invasion and self-renewal capacity, and CSC surface markers expression, with alterations of β-catenin in pancreatic cancer stem-like cells. The combination of quercetin and gemcitabine can reduce tumor growth and decrease drug resistance in pancreatic cancer. β-Catenin plays an important role in maintenance and progression of pancreatic cancer. Targeting β-catenin using quercetin combined with gemcitabine may be a treatment strategy to improve prognosis in patients with pancreatic cancer.

Effective photothermal chemotherapy with docetaxel-loaded gold nanospheres in advanced prostate cancer

Background: Multifunctional gold nanospheres (MGNs)-loaded with docetaxel (MGN@DTX) were prepared and evaluated for therapeutic efficacy in nude mice bearing human prostate cancer xenografts.Methods: MGNs were prepared from PEGylated hollow gold nanospheres (HGNs) coated with folic acid and DTPTT chelate. Then, the effect of radiolabelled MGNs (99mTc-MGNs) on PC-3 cell apoptosis was assessed by flow cytometry, while their binding affinity to these cells was evaluated by cell binding assays. Next, biodistribution of 99mTc-MGNs in xenograft bearing mice was measured by SPECT imaging. Also, DTX loading and release rates were estimated in MGN@DTX. Finally, in vitro stability in human serum and cytotoxicity of MGN@DTX were assessed, as well as their antitumor effect in xenograft bearing mice.Results: 99mTc-MGNs (97.69% purity) showed good binding affinity to PC-3 cells, a specific recognition blocked by excess folic acid. Interestingly, MGN@DTX remained stable in human serum for 24 h, and exhibited higher mean cytotoxicity after NIR laser irradiation than free DTX. By day 28, tumor inhibition rates were higher in the MGN@DTX + NIR laser irradiation group compared with the DTX and MGNs + NIR laser irradiation groups.Conclusions: Loading chemotherapeutic drugs into MGNs can increase antitumor potency, reduce normal cell damage and decrease drug resistance, thus representing a promising approach for advanced prostate cancer treatment.

Downregulation of MACC1 expression enhances cisplatin sensitivity in SKOV-3/DDP cells.

The aim of this study was to investigate the correlation between MACC1 expression and resistance to cisplatin (DDP) in DDP-resistant human epithelial ovarian cancer SKOV-3 cells (SKOV-3/DDP). MACC1 mRNA and protein expression levels in SKOV-3 and SKOV-3/DDP cells were detected by reverse transcriptase polymerase chain reaction and western blot. The SKOV-3/DDP cells were divided into 5 groups: control, shVect (transfected with p-super-EGFP-1 plasmid), pshMACC1 (transfected with psuper-EGFP-shMACC1 plasmid), PD (pretreated with 20 μM PD98059), and combined (transfected with psuper-EGFP-shMACC1 plasmid and pretreated with 20 μM PD98059) groups. Cisplatin sensitivity and cell apoptosis in SKOV-3/DDP cells were assayed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and flow cytometry. ERK1/2 and p-ERK1/2 expression was determined by western blot. MACC1 mRNA and protein expression levels in SKOV-3/DDP cells were 2.66 ± 0.54 and 1.95 ± 0.45 times those seen in SKOV-3 cells (P < 0.05). Cisplatin sensitivity of pshMACC1 group was much higher than that in the control and shVect groups. Cisplatin-induced cell apoptosis rates increased significantly in the pshMACC1, PD, and combined groups, compared to the control and shVect groups. Moreover, the apoptosis rate was the highest in the combined group among the 5 groups (IC50 = 20.836 ± 0.629 μM). p-ERK1/2 expression decreased significantly in the pshMACC1, PD, and combined groups (this decrease was the most obvious in the combined group). In conclusion, downregulation of MACC1 expression could enhance cisplatin sensitivity and decrease drug resistance in SKOV- 3/DDP cells.

Crystal Structure of the Human Fatty Acid Synthase Enoyl-Acyl Carrier Protein-Reductase Domain Complexed with Triclosan Reveals Allosteric Protein-Protein Interface Inhibition

Human fatty acid synthase (FAS) is a large, multidomain protein that synthesizes long chain fatty acids. Because these fatty acids are primarily provided by diet, FAS is normally expressed at low levels; however, it is highly up-regulated in many cancers. Human enoyl-acyl carrier protein-reductase (hER) is one of the FAS catalytic domains, and its inhibition by drugs like triclosan (TCL) can increase cytotoxicity and decrease drug resistance in cancer cells. We have determined the structure of hER in the presence and absence of TCL. TCL was not bound in the active site, as predicted, but rather at the protein-protein interface (PPI). TCL binding induces a dimer orientation change that causes downstream structural rearrangement in critical active site residues. Kinetics studies indicate that TCL is capable of inhibiting the isolated hER domain with an IC50 of ∼ 55 μM. Given the hER-TCL structure and the inhibition observed in the hER domain, it seems likely that TCL is observed in the physiologically relevant binding site and that it acts as an allosteric PPI inhibitor. TCL may be a viable scaffold for the development of anti-cancer PPI FAS inhibitors.

Correlation between use of antiretroviral adherence devices by HIV-infected youth and plasma HIV RNA and self-reported adherence.

Our objective was to investigate antiretroviral adherence device use by HIV-infected youth and assess associations of device use with viral suppression and self-reported adherence. This cross-sectional, multisite, clinic-based study included data from 1,317 HIV-infected individuals 12-24 years of age that were prescribed antiretroviral therapy. Mean adherence in the past 7 days was 86.1 % and 50.5 % had an undetectable HIV RNA. Pillbox was the most commonly endorsed device. No specific device was independently associated with higher odds of 100 % adherence. Paradoxically, having an undetectable HIV RNA was inversely associated with use of adherence devices (OR 0.80; p = 0.04); however, among those with <100 % adherence, higher adherence was associated with use of one or more adherence devices (coefficient = 7.32; p = 0.003). Our data suggest that adolescents who experienced virologic failure often used adherence devices which may not have been sufficiently effective in optimizing adherence. Therefore, other tailored adherence-enhancing methods need to be considered to maximize virologic suppression and decrease drug resistance and HIV transmission.

The effect and mechanism of vinorelbine on cisplatin resistance of human lung cancer cell line A549/DDP

背景与目的肺癌细胞耐药已经成为肺癌化疗的主要困难之一，异长春花碱被认为可有效抑制肺癌细胞的增殖和转移。本研究旨在探讨异长春花碱对人肺癌A549/DDP细胞顺铂耐受性的逆转作用及机制。方法1 μmol/L和5 μmol/L异长春花碱作用A549/DDP细胞后，应用MTS法检测肿瘤细胞顺铂敏感性的变化，应用流式细胞术检测肿瘤细胞凋亡率变化，肿瘤细胞对Rh-123摄入量的变化，Western blot法检测MDR1、Bcl-2、survivin、caspase-3/8和PTEN蛋白表达以及Akt的磷酸化水平的变化，real-time PCR检测MDR1、Bcl-2、survivin和PTEN的mRNA表达，用报告基因系统检测NF-κB、Twist和Snail的转录活性。结果1 μmol/L和5 μmol/L异长春花碱作用A549/DDP细胞后，肿瘤细胞对顺铂的敏感性分别提高了1.91倍和2.54倍，肿瘤细胞对Rh-123的摄入量提高了1.93倍和2.95倍，细胞凋亡增加了2.25倍和3.82倍，MDR1、Bcl-2、survivin蛋白表达和Akt磷酸化水平下调，caspase-3/8和PTEN蛋白表达上调，MDR1的mRNA表达下调43.5%和25.8%，Bcl-2的mRNA表达下调57.3%和34.1%，survivin的mRNA表达下调37.6%和12.4%，PTEN表达上调183.4%和154.2%，NF-κB转录活性下降53.2%和34.5%，Twist转录活性下降61.4%和33.5%，Snail转录活性下降57.8%和18.7%。结论异长春花碱可提高肿瘤细胞A549/DDP对顺铂的敏感性，其机制可能与调节PTEN/AKT/NF-κB信号路径活性，进而下调耐药基因表达，上调促凋亡基因表达有关。

Expansion of HAART Coverage Is Associated with Sustained Decreases in HIV/AIDS Morbidity, Mortality and HIV Transmission: The “HIV Treatment as Prevention” Experience in a Canadian Setting

BackgroundThere has been renewed call for the global expansion of highly active antiretroviral therapy (HAART) under the framework of HIV treatment as prevention (TasP). However, population-level sustainability of this strategy has not been characterized.MethodsWe used population-level longitudinal data from province-wide registries including plasma viral load, CD4 count, drug resistance, HAART use, HIV diagnoses, AIDS incidence, and HIV-related mortality. We fitted two Poisson regression models over the study period, to relate estimated HIV incidence and the number of individuals on HAART and the percentage of virologically suppressed individuals.ResultsHAART coverage, median pre-HAART CD4 count, and HAART adherence increased over time and were associated with increasing virological suppression and decreasing drug resistance. AIDS incidence decreased from 6.9 to 1.4 per 100,000 population (80% decrease, p = 0.0330) and HIV-related mortality decreased from 6.5 to 1.3 per 100,000 population (80% decrease, p = 0.0115). New HIV diagnoses declined from 702 to 238 cases (66% decrease; p = 0.0004) with a consequent estimated decline in HIV incident cases from 632 to 368 cases per year (42% decrease; p = 0.0003). Finally, our models suggested that for each increase of 100 individuals on HAART, the estimated HIV incidence decreased 1.2% and for every 1% increase in the number of individuals suppressed on HAART, the estimated HIV incidence also decreased by 1%.ConclusionsOur results show that HAART expansion between 1996 and 2012 in BC was associated with a sustained and profound population-level decrease in morbidity, mortality and HIV transmission. Our findings support the long-term effectiveness and sustainability of HIV treatment as prevention within an adequately resourced environment with no financial barriers to diagnosis, medical care or antiretroviral drugs. The 2013 Consolidated World Health Organization Antiretroviral Therapy Guidelines offer a unique opportunity to further evaluate TasP in other settings, particularly within generalized epidemics, and resource-limited setting, as advocated by UNAIDS.

The Mechanism of DAB2IP in Chemoresistance of Prostate Cancer Cells

The docetaxel-based chemotherapy is the standard of care for castration-resistant prostate cancer (CRPC), inevitably, patients develop resistance and decease. Until now, the mechanism and predictive marker for chemoresistance are poorly understood. Immortalized normal prostate and cancer cell lines stably manipulated with different DAB2IP expression levels were used and treated with chemotherapeutic drugs commonly used in prostate cancer therapy. Cell proliferation was measured using MTT assay; Western blot, quantitative PCR, and luciferase reporter assays were used to analyze Clusterin gene regulation by DAB2IP. Immunohistochemical analysis was conducted for evaluating DAB2IP, Clusterin and Egr-1 expression in human prostate cancer tissue. DAB2IP Knockdown (KD) cells exhibited resistance to several chemotherapeutic drugs, whereas increased DAB2IP in C4-2 cells restored the drug sensitivity. Parallel, DAB2IP KD cells exhibited higher expression of Clusterin, an antiapoptotic factor, whereas elevated DAB2IP in C4-2 cells decreased Clusterin expression. Functionally, knocking down Clusterin by short-hairpin RNA or antisense oligonucleotide OGX-011 decreased drug resistance, whereas overexpressing Clusterin in C4-2 D2 enhanced drug resistance. Mechanistically, DAB2IP blocked the cross-talk between Wnt/β-catenin and IGF-I signaling, leading to the suppression of Egr-1 that is responsible for Clusterin expression. A similar result was observed in the prostate of DAB2IP knockout animals. In addition, we observed a significantly inverse correlation between DAB2IP and Egr-1 or Clusterin expression from clinical tissue microarray. This study unveils a new regulation of the Egr-1/Clusterin signaling network by DAB2IP. Loss of DAB2IP expression in CRPC cells signifies their chemoresistance. Clusterin is a key target for developing more effective CRPC therapy.

Aldose reductase inhibition enhances TRAIL-induced human colon cancer cell apoptosis through AKT/FOXO3a-dependent upregulation of death receptors

One of the major problems associated with the chemotherapy of tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) that selectively kills tumor cells is decreased drug resistance. This warranted the development of safe novel pharmacological agents that could sensitize the tumor cells to TRAIL. Herein, we examined the role of aldose reductase (AR) in sensitizing cancer cells to TRAIL and potentiating TRAIL-induced apoptosis of human colon cancer cells. We demonstrate that AR inhibition potentiates TRAIL-induced cytotoxicity in cancer cells by upregulation of both death receptor (DR)-5 and DR4. Knockdown of DR5 and DR4 significantly (>85%) reduced the sensitizing effect of the AR inhibitor fidarestat on TRAIL-induced apoptosis. Further, AR inhibition also downregulates cell survival proteins (Bcl-xL, Bcl-2, survivin, XIAP, and FLIP) and upregulates the expression of proapoptotic proteins such as Bax and alters mitochondrial membrane potential, leading to cytochrome c release, caspases-3 activation, and PARP cleavage. We found that AR inhibition regulates AKT/PI3K-dependent activation of forkhead transcription factor FOXO3a. Knockdown of FOXO3a significantly (>80%) abolished AR inhibition-induced upregulation of DR5 and DR4 and apoptosis in colon cancer cells. Overall, our results show that fidarestat potentiates TRAIL-induced apoptosis through downregulation of cell survival proteins and upregulation of death receptors via activation of the AKT/FOXO3a pathway.

Abstract 2920: Effect of nitric oxide on invasiveness and metabolism of cancer cells.

Abstract Nitric oxide synthase (NOS) expression has been shown to be upregulated in cancer cells compared to normal cells. The resulting increased nitric oxide (NO) production changes the metastatic ability of cancer cells. The link between NOS expression, NO production, apoptosis, and drug resistance in ovarian cancer is beginning to be studied. However, the effect of NO on metabolism and invasiveness of ovarian cancer cells is not known. Here, using gas chromatography - mass spectroscopy based isotope analysis we show that highly-invasive ovarian cancer cells convert arginine to citrulline via the nitric oxide pathway as opposed to less-invasive ovarian cancer cells. We hypothesize that NO is important for the proliferation, migration, invasion, and drug resistance in highly-invasive ovarian cancer cells as opposed to less-invasive ovarian cancer cells, and that these changes in metastatic behavior are linked to changes in metabolism. We show that L-NG-Nitroarginine Methyl Ester (L-NAME), an inhibitor of NOS, decreases NO production and thus decreases proliferation, migration, and invasion of ovarian cancer cells, as well as decreases drug resistance. Using metabolic assays, we show that NO affects the glucose consumption, lactate secretion, and pyruvate uptake, and provides us with clues regarding the effect of NO on glycolytic activity of highly-invasive and less-invasive ovarian cancer cells. Lastly, we show that NO affects oxygen consumption rate in these cells, and thus oxidative phosphorylation. Our findings are the first to link NO production and invasiveness with metabolism in ovarian cancer cells. Citation Format: Christine A. Caneba, Juan Marini, Deepak Nagrath. Effect of nitric oxide on invasiveness and metabolism of cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2920. doi:10.1158/1538-7445.AM2013-2920

Latest approved therapies for metastatic melanoma: what comes next?

Nowadays, oncogene-directed therapy and immunotherapy represent the two most promising avenues for patients with metastatic melanoma. The recent oncogene-directed therapeutic, vemurafenib, usually produces high level of tumor shrinkage and survival benefits in many patients with B-RAF V600E mutant melanomas, although the fast and high degrees of responses are likely short-lived. Conversely, the newly-approved immunotherapeutic, ipilimumab, produces durable responses in patients presenting CTLA-4 T-cell surface protein. Nevertheless, the possible synergy in combining these two therapeutic strategies primarily rely on the rational design of medical protocols (e.g., sequence and timing of agent administration; drug selectivity; compatibility of combined therapies i.e., adoptive T cell or agents, i.e., MEK inhibitor trametinib, PD-1 and PDL-1 blockers). Improved therapeutic protocols shall overcome therapeutic limitations such as the (i) tolerability and safety (i.e., minimal toxic side-effects); (ii) progression free survival (e.g., reduced relapse disease frequency); (iii) duration response (i.e., decreased drug resistance). Eventually, multidisciplinary approaches are still requested (e.g., genomics for personalized medicine, nanomedicine to overcome low free-drug bioavailability and targeting, systematic search of “melanoma stem cells” to enhance the prognosis and develop more valuable theranostics). In this paper, I will mainly present and discuss the latest and promising treatments for advanced cutaneous melanomas.

Applications of nanotechnology for melanoma treatment, diagnosis, and theranostics

Melanoma is the most aggressive type of skin cancer and has very high rates of mortality. An early stage melanoma can be surgically removed, with a survival rate of 99%. However, metastasized melanoma is difficult to cure. The 5-year survival rates for patients with metastasized melanoma are still below 20%. Metastasized melanoma is currently treated by chemotherapy, targeted therapy, immunotherapy and radiotherapy. The outcome of most of the current therapies is far from optimistic. Although melanoma patients with a mutation in the oncogene v-Raf murine sarcoma viral oncogene homolog B1 (BRAF) have an initially higher positive response rate to targeted therapy, the majority develop acquired drug resistance after 6 months of the therapy. To increase treatment efficacy, early diagnosis, more potent pharmacological agents, and more effective delivery systems are urgently needed. Nanotechnology has been extensively studied for melanoma treatment and diagnosis, to decrease drug resistance, increase therapeutic efficacy, and reduce side effects. In this review, we summarize the recent progress on the development of various nanoparticles for melanoma treatment and diagnosis. Several common nanoparticles, including liposome, polymersomes, dendrimers, carbon-based nanoparticles, and human albumin, have been used to deliver chemotherapeutic agents, and small interfering ribonucleic acids (siRNAs) against signaling molecules have also been tested for the treatment of melanoma. Indeed, several nanoparticle-delivered drugs have been approved by the US Food and Drug Administration and are currently in clinical trials. The application of nanoparticles could produce side effects, which will need to be reduced so that nanoparticle-delivered drugs can be safely applied in the clinical setting.

The Plasma Membrane Sialidase NEU3 Regulates the Malignancy of Renal Carcinoma Cells by Controlling β1 Integrin Internalization and Recycling

The human plasma membrane sialidase NEU3 is a key enzyme in the catabolism of membrane gangliosides, is crucial in the regulation of cell surface processes, and has been demonstrated to be significantly up-regulated in renal cell carcinomas (RCCs). In this report, we show that NEU3 regulates β1 integrin trafficking in RCC cells by controlling β1 integrin recycling to the plasma membrane and controlling activation of the epidermal growth factor receptor (EGFR) and focal adhesion kinase (FAK)/protein kinase B (AKT) signaling. NEU3 silencing in RCC cells increased the membrane ganglioside content, in particular the GD1a content, and changed the expression of key regulators of the integrin recycling pathway. In addition, NEU3 silencing up-regulated the Ras-related protein RAB25, which directs internalized integrins to lysosomes, and down-regulated the chloride intracellular channel protein 3 (CLIC3), which induces the recycling of internalized integrins to the plasma membrane. In this manner, NEU3 silencing enhanced the caveolar endocytosis of β1 integrin, blocked its recycling and reduced its levels at the plasma membrane, and, consequently, inhibited EGFR and FAK/AKT. These events had the following effects on the behavior of RCC cells: they (a) decreased drug resistance mediated by the block of autophagy and the induction of apoptosis; (b) decreased metastatic potential mediated by down-regulation of the metalloproteinases MMP1 and MMP7; and (c) decreased adhesion to collagen and fibronectin. Therefore, our data identify NEU3 as a key regulator of the β1 integrin-recycling pathway and FAK/AKT signaling and demonstrate its crucial role in RCC malignancy.

Identification and Phenotypic Characterization of a Subpopulation of Acute Myelogenous Leukemia (AML) Cells with Increased Plastic Adherence.

Abstract 2556The high incidence of relapse in acute myelogenous leukemia (AML) patients has been attributed to the existence of a small population of leukemic stem cells (LSC) that current therapies are unable to eradicate. LSC, in similarity to normal hematopoietic stem cells (HSC), are able to engraft, self-renew, and interact with cells within the bone marrow (BM) niche. During leukemogenesis, changes within the BM microenvironment promote LSC survival and expansion, and shelter leukemic cells from chemotherapy. Therefore, displacing these cells from the BM niches prior to chemotherapy may decrease drug resistance and prevent relapse of the disease. The binding of α4β1 integrins (VLA-4 or CD49d/CD29) to the stromal extracellular matrix and cell surface ligands is a key component in homing and trafficking, specifically in the migration of HSC beneath marrow stromal cells, and VLA-4 has been shown to be crucial for the persistence of minimal residual disease in AML. KG1a is a differentiation-resistant AML cell line containing a very primitive population of CD34+CD38- cells. Upon culture, the majority of KG1a cells remain in suspension, while a small percentage adheres to the tissue culture plastic (Adh-KG1a). Here, we hypothesized that characterization of adhesion molecules that were either unique or significantly altered in this subset could lead to identification of putative therapeutic targets for dislodging AML cells from microenvironmental niches. KG1a and Adh-KG1a populations were evaluated by flow-cytometry for the presence of CD49d and CD29.The FACSort results were then analyzed using FlowJo7.6 software. No statistically significant (p>0.05) differences were found in the percentage of Adh-KG1a and KG1a that expressed CD49d (91.6±3% vs. 86.2±5%) or CD29 (95.0±2% vs. 91.8±1.8%). However, we found that there is a small and unique population of adherent CD29+ cells, with lower fluorescent intensity (MFI=326), that is not present in the non-adherent population (MFI=435), suggesting that this low level of surface expression is unique to the adherent fraction. Analysis of CD11a, CD44, CD18, CD106, CD105, CD34, CD107 and CD38 showed that none of these molecules were expressed at significantly different levels between KG1a and the Adh-KG1a fraction. Addition of anti-CD44 or anti-CD29 antibodies to the cells in culture did not result in decreased numbers of Adh-KG1a cells/flask, but resulted in induction of heterotypic aggregation of Adh-KG1a. Cell cycle analysis did not show significant differences between the 2 populations. However, analysis of CD54 expression demonstrated that 12.2% more of the Adh-KG1a were positive for CD54 than the non-adherent cells. Furthermore, characterization of gangliosides on KG1a and Adh-KG1a showed that the non-adherent fraction contained significantly more GM3 than the Adh-KG1a. Given prior reports showing that: 1) high levels of VLA-4 (CD49d/CD29) are associated with better prognosis in AML; 2) high levels of CD54 correlate with low relapse-free survival probability in AML; and 3) ganglioside composition has been shown to exert a pronounced effect on drug-resistance/sensitivity in AML, our findings of an adherent population of primitive hematopoietic stem/progenitor cells within KG1a that are CD29dim, express high levels of CD54, and contain lower levels of GM3, suggest that this unique subpopulation may play an important role in relapse in AML, and could represent a target for novel therapeutics that could better eradicate the elusive LSC and promise an improved disease-free survival rate in AML. Disclosures:No relevant conflicts of interest to declare.

Quercetin Suppresses Drug-Resistant Spheres via the p38 MAPK–Hsp27 Apoptotic Pathway in Oral Cancer Cells

BackgroundTreatment failure in oral squamous cell carcinoma (OSCC) leading to local recurrence(s) and metastases is mainly due to drug resistance. Cancer stem cells (CSCs) are thought be responsible for the development of drug resistance. However, the correlations between CSCs, drug resistance, and new strategy against drug resistance in OSCC remain elusive.MethodsA drug-resistant sphere (DRSP) model was generated by using a nonadhesive culture system to induce drug-resistant cells from SCC25 oral cancer cells. A comparative analysis was performed between the parent control cells and DRSPs with a related treatment strategy focusing on the expression of epithelial–mesenchymal transition (EMT)-associated markers, drug-resistance-related genes, and CSC properties in vitro, as well as tumorigenicity and the regimen for tumor regression in vivo.ResultsOur data show the presence of a phenomenon of EMT with gradual cellular transition from an epithelioid to mesenchymal-like spheroid morphology during induction of drug resistance. The characterization of DRSPs revealed the upregulation of the drug-resistance-related genes ABCG2 and MDR-1 and of CSC-representative markers, suggesting that DRSPs have greater resistance to cisplatin (Cis) and stronger CSC properties compared with the control. Moreover, overexpression of phosphorylated heat-shock protein 27 (p-Hsp27) via the activation of p38 MAPK signaling was observed in DRSPs. Knockdown of Hsp27 decreased Cis resistance and induced apoptosis in DRSPs. Furthermore, an inhibitor of Hsp27, quercetin (Qu), suppressed p-Hsp27 expression, with alterations of the EMT signature, leading to the promotion of apoptosis in DRSPs. A xenographic study also confirmed the increase of tumorigenicity in DRSPs. The combination of Qu and Cis can reduce tumor growth and decrease drug resistance in OSCC.ConclusionsThe p38 MAPK–Hsp27 axis plays an important role in CSCs-mediated drug resistance in OSCC. Targeting this axis using Qu combined with Cis may be a treatment strategy to improve prognosis in patients with OSCC.