Prognostication of Soft Tissue Sarcomas Based on Chromosome 17q Gene and Protein Status: Evaluation of TOP2A, HER-2/neu, and Survivin

Topoisomerase 2 alpha (), HER-2/ and are genes that lie on chromosome 17 and correlate with the prognosis and prediction of target-driven therapy against tumors. In a previous study, we showed that TOP2A transcripts levels were significantly higher in soft tissue sarcomas (STS) than in benign tumors and desmoid-type fibromatoses (FM). Because these genes have been insufficiently examined in STS, we aimed to identify alterations in TOP2A and HER-2 expression by fluorescent in situ hybridization and immunohistochemistry, as well as that of survivin, and correlate them with clinicopathologic findings to assess their prognostic value. Eighteen FM and 244 STS were included. Fluorescent in situ hybridization and immunohistochemistry were performed on a tissue microarray. TOP2A and survivin were more highly expressed in sarcomas than in FM. TOP2A was an independent predictor of an unfavorable prognosis; it was combined with formerly established prognostic factors (primarily histologic grade and tumor size at diagnosis) to create a prognostic index that evaluated overall survival. Gene amplification/polysomy (13%) did not correlate with protein overexpression. Survivin and HER-2 expression were not associated with patient outcomes. These findings might become valuable in the management of patients with STS and possibly in the prospective evaluation of responses to new target-driven therapies.

New mutations in the GLA gene in Brazilian families with Fabry disease

Fabry disease (FD) is an X-linked inborn error of glycosphingolipid catabolism that results from mutations in the alpha-galactosidase A (GLA) gene. Evaluating the enzymatic activity in male individuals usually performs the diagnosis of the disease, but in female carriers the diagnosis based only on enzyme assays is often inconclusive. In this work, we analyzed 568 individuals from 102 families with suspect of FD. Overall, 51 families presented 38 alterations in the GLA gene, among which 19 were not previously reported in literature. The alterations included 17 missense mutations, 7 nonsense mutations, 7 deletions, 6 insertions and 1 in the splice site. Six alterations (R112C, R118C, R220X, R227X, R342Q and R356W) occurred at CpG dinucleotides. Five mutations not previously described in the literature (A156D, K237X, A292V, I317S, c.1177_1178insG) were correlated with low GLA enzyme activity and with prediction of molecular damages. From the 13 deletions and insertions, 7 occurred in exons 6 or 7 (54%) and 11 led to the formation of a stop codon. The present study highlights the detection of new genomic alterations in the GLA gene in the Brazilian population, facilitating the selection of patients for recombinant enzyme-replacement trials and offering the possibility to perform prenatal diagnosis. Journal of Human Genetics (2012) 57, 347-351; doi:10.1038/jhg.2012.32; published online 3 May 2012

Atorvastatin and hormone therapy effects on APOE mRNA expression in hypercholesterolemic postmenopausal women

Menopause is associated with changes in lipid levels resulting in increased risk of atherosclerosis and cardiovascular events. Hormone therapy (HT) and atorvastatin have been used to improve lipid profile in postmenopausal women. Effects of HT, atorvastatin and APOE polymorphisms on serum lipids and APOE and LXRA expression were evaluated in 87 hypercholesterolemic postmenopausal women, randomly selected for treatment with atorvastatin (AT, n=17), estrogen or estrogen plus progestagen (HT, n=34) and estrogen or estrogen plus progestagen associated with atorvastatin (HT+AT, n=36). RNA was extracted from peripheral blood mononuclear cells (PBMC) and mRNA expression was measured by TaqMan (R) PCR. APOE epsilon 2/epsilon 3/epsilon 4 genotyping was performed using PCR-RFLP. Total cholesterol (TC). LDL-c and apoB were reduced after each treatment (p<0.001). Triglycerides, VLDL-c and apoAl were reduced only after atorvastatin (p<0.05), whereas triglycerides and VLDL-c were increased after HT (p=0.01). HT women had lower reduction on TC, LDL-c and apoB than AT and HT+AT groups (p<0.05). APOE mRNA expression was reduced after atorvastatin treatment (p=0.03). Although LXRA gene expression was not modified by atorvastatin, it was correlated with APOE mRNA before and after treatments. Basal APOE mRNA expression was not influenced by gene polymorphisms, however the reduction on APOE expression was more pronounced in epsilon 3 epsilon 3 than in epsilon 3 epsilon 4 carriers. Atorvastatin down-regulates APOE mRNA expression and it is modified by APOE genotypes in PBMC from postmenopausal women. (C) 2011 Elsevier Ltd. All rights reserved.

Low-level laser therapy (808 nm) reduces inflammatory response and oxidative stress in rat tibialis anterior muscle after cryolesion

Background and Objective Muscle regeneration is a complex phenomenon, involving coordinated activation of several cellular responses. During this process, oxidative stress and consequent tissue damage occur with a severity that may depend on the intensity and duration of the inflammatory response. Among the therapeutic approaches to attenuate inflammation and increase tissue repair, low-level laser therapy (LLLT) may be a safe and effective clinical procedure. The aim of this study was to evaluate the effects of LLLT on oxidative/nitrative stress and inflammatory mediators produced during a cryolesion of the tibialis anterior (TA) muscle in rats. Material and Methods Sixty Wistar rats were randomly divided into three groups (n?=?20): control (BC), injured TA muscle without LLLT (IC), injured TA muscle submitted to LLLT (IRI). The injured region was irradiated daily for 4 consecutive days, starting immediately after the lesion using a AlGaAs laser (continuous wave, 808?nm, tip area of 0.00785?cm2, power 30?mW, application time 47?seconds, fluence 180?J/cm2; 3.8?mW/cm2; and total energy 1.4?J). The animals were sacrificed on the fourth day after injury. Results LLLT reduced oxidative and nitrative stress in injured muscle, decreased lipid peroxidation, nitrotyrosine formation and NO production, probably due to reduction in iNOS protein expression. Moreover, LLLT increased SOD gene expression, and decreased the inflammatory response as measured by gene expression of NF-k beta and COX-2 and by TNF-a and IL-1 beta concentration. Conclusion These results suggest that LLLT could be an effective therapeutic approach to modulate oxidative and nitrative stress and to reduce inflammation in injured muscle. Lasers Surg. Med. 44: 726735, 2012. (c) 2012 Wiley Periodicals, Inc.

DNA vaccine containing the mycobacterial hsp65 gene prevented insulitis in MLD-STZ diabetes

Abstract Background Our group previously demonstrated that a DNA plasmid encoding the mycobacterial 65-kDa heat shock protein (DNA-HSP65) displayed prophylactic and therapeutic effect in a mice model for tuberculosis. This protection was attributed to induction of a strong cellular immunity against HSP65. As specific immunity to HSP60 family has been detected in arthritis, multiple sclerosis and diabetes, the vaccination procedure with DNA-HSP65 could induce a cross-reactive immune response that could trigger or worsen these autoimmune diseases. Methods In this investigation was evaluated the effect of a previous vaccination with DNA-HSP65 on diabetes development induced by Streptozotocin (STZ). C57BL/6 mice received three vaccine doses or the corresponding empty vector and were then injected with multiple low doses of STZ. Results DNA-HSP65 vaccination protected mice from STZ induced insulitis and this was associated with higher production of IL-10 in spleen and also in the islets. This protective effect was also concomitant with the appearance of a regulatory cell population in the spleen and a decreased infiltration of the islets by T CD8+ lymphocytes. The vector (DNAv) also determined immunomodulation but its protective effect against insulitis was very discrete. Conclusion The data presented in this study encourages a further investigation in the regulatory potential of the DNA-HSP65 construct. Our findings have important implications for the development of new immune therapy strategies to combat autoimmune diseases.

Tissue distribution of a plasmid DNA encoding Hsp65 gene is dependent on the dose administered through intramuscular delivery

In order to assess a new strategy of DNA vaccine for a more complete understanding of its action in immune response, it is important to determine the in vivo biodistribution fate and antigen expression. In previous studies, our group focused on the prophylactic and therapeutic use of a plasmid DNA encoding the Mycobacterium leprae 65-kDa heat shock protein (Hsp65) and achieved an efficient immune response induction as well as protection against virulent M. tuberculosis challenge. In the present study, we examined in vivo tissue distribution of naked DNA-Hsp65 vaccine, the Hsp65 message, genome integration and methylation status of plasmid DNA. The DNA-Hsp65 was detectable in several tissue types, indicating that DNA-Hsp65 disseminates widely throughout the body. The biodistribution was dose-dependent. In contrast, RT-PCR detected the Hsp65 message for at least 15 days in muscle or liver tissue from immunized mice. We also analyzed the methylation status and integration of the injected plasmid DNA into the host cellular genome. The bacterial methylation pattern persisted for at least 6 months, indicating that the plasmid DNA-Hsp65 does not replicate in mammalian tissue, and Southern blot analysis showed that plasmid DNA was not integrated. These results have important implications for the use of DNA-Hsp65 vaccine in a clinical setting and open new perspectives for DNA vaccines and new considerations about the inoculation site and delivery system.

Independent of ErbB1 gene copy number, EGF stimulates migration but is not associated with cell proliferation in non-small cell lung cancer

Abstract Background Lung cancer often exhibits molecular changes, such as the overexpression of the ErbB1 gene. ErbB1 encodes epidermal growth factor receptor (EGFR), a tyrosine kinase receptor, involved mainly in cell proliferation and survival. EGFR overexpression has been associated with more aggressive disease, poor prognosis, low survival rate and low response to therapy. ErbB1 amplification and mutation are associated with tumor development and are implicated in ineffective treatment. The aim of the present study was to investigate whether the ErbB1 copy number affects EGFR expression, cell proliferation or cell migration by comparing two different cell lines. Methods The copies of ErbB1 gene was evaluated by FISH. Immunofluorescence and Western blotting were performed to determine location and expression of proteins mentioned in the present study. Proliferation was studied by flow cytometry and cell migration by wound healing assay and time lapse. Results We investigated the activation and function of EGFR in the A549 and HK2 lung cancer cell lines, which contain 3 and 6 copies of ErbB1, respectively. The expression of EGFR was lower in the HK2 cell line. EGFR was activated after stimulation with EGF in both cell lines, but this activation did not promote differences in cellular proliferation when compared to control cells. Inhibiting EGFR with AG1478 did not modify cellular proliferation, confirming previous data. However, we observed morphological alterations, changes in microfilament organization and increased cell migration upon EGF stimulation. However, these effects did not seem to be consequence of an epithelial-mesenchymal transition. Conclusion EGFR expression did not appear to be associated to the ErbB1 gene copy number, and neither of these aspects appeared to affect cell proliferation. However, EGFR activation by EGF resulted in cell migration stimulation in both cell lines.

Periodontopathogens levels and clinical response to periodontal therapy in individuals with the interleukin-4 haplotype associated with susceptibility to chronic periodontitis

Periodontitis is an inflammatory disease that results from an interaction between dental biofilm agents and the host immune-inflammatory response. Periodontopathogenic organisms, such as Porphyromonas gingivalis, Tannerella forsythia, and Treponema denticola, as well as the host's susceptibility, represented by the host's genetic makeup, are the key factors that influence this complex disease. Recently, we identified haplotypes in the IL4 gene that were associated with chronic periodontitis (CP). This study aimed to evaluate whether subjects with different IL4 haplotypes (TCI/CCI and TTD/CTI) would be differentially colonized by periodontopathogens and whether they would respond differently to non-surgical periodontal therapy. Thirty-nine patients carrying the IL4 haplotype of genetic susceptibility to CP (IL4+) or protection against CP (IL4-) were evaluated. Those groups were further subdivided into individuals with CP (CP IL4+ or CP IL4-) and those that were periodontally healthy (H) (H IL4+ or H IL4-). CP patients were submitted to non-surgical periodontal therapy. Clinical and microbiological analyses were performed considering the data at baseline and 45 and 90 days after periodontal therapy. Periodontopathogens levels were evaluated by absolute quantitative polymerase chain reaction (qPCR). The baseline data revealed that the total levels of periodontopathogens were higher in the CP IL4+ than in the CP IL4- groups. Clinical analyses revealed that the periodontal therapy was equally effective, independent of the subject's IL4 genetic load. The TCI/CCI IL4 haplotype, previously associated with genetic susceptibility to CP, was also associated with increased levels of periodontopathogenic bacteria, but this genetic background did not influence the response to non-surgical periodontal treatment.

Morphological alterations and gene and protein expression profiling of bladder tumor cells after treatment with gemcitabine.

Chemical agents used in cancer therapy are associated with cell cycle arrest, activation or deactivation of mechanisms associated to DNA repair and apoptosis. However, due to the complexity of biological systems, the molecular mechanisms responsible for these activities are not fully understood. Thus, studies about gene and protein expression have shown promising results for understanding the mechanisms related to cellular responses and regression of cancer after chemotherapy. This study aimed to evaluate the gene and protein expression profiling in bladder transitional cell carcinoma (TCC) with different TP53 status after gemcitabine (1.56 μM) treatment. The RT4 (grade 1, TP53 wild type), 5637 (grade 2, TP53 mutated) and T24 (grade 3, TP53 mutated) cell lines were used. PCR arrays and mass spectrometry were used to analyze gene and protein expression, respectively. Morphological alterations were observed using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The results of PCR array showed that gemcitabine activity was mainly related to CDKN1A, GADD45A and SERTDA1 overexpression, and BAX overexpression only in the wild type TP53 cells. Mass spectrometry demonstrated that gemcitabine modulated the protein expression, especially those from genes related to apoptosis, transport of vesicles and stress response. Analyses using SEM and TEM showed changes in cell morphology independently on the cell line studied. The observed decreased number of microvillus suggests low contact among the cells and between cell and extracellular matrix; irregular forms might indicate actin cytoskeleton deregulation; and the reduction in the amount of organelles and core size might indicate reduced cellular metabolism. In conclusion, independently on TP53 status or grade of bladder tumor, gemcitabine modulated genes related to the cell cycle and apoptosis, that reflected in morphological changes indicative of future cell death.

Exome analysis of HIV patients submitted to dendritic cells therapeutic vaccine reveals an association of CNOT1 gene with response to the treatment

INTRODUCTION: With the aim of searching genetic factors associated with the response to an immune treatment based on autologous monocyte-derived dendritic cells pulsed with autologous inactivated HIV, we performed exome analysis by screening more than 240,000 putative functional exonic variants in 18 HIV-positive Brazilian patients that underwent the immune treatment. METHODS: Exome analysis has been performed using the ILLUMINA Infinium HumanExome BeadChip. zCall algorithm allowed us to recall rare variants. Quality control and SNP-centred analysis were done with GenABEL R package. An in-house implementation of the Wang method permitted gene-centred analysis. RESULTS: CCR4-NOT transcription complex, subunit 1 (CNOT1) gene (16q21), showed the strongest association with the modification of the response to the therapeutic vaccine (p=0.00075). CNOT1 SNP rs7188697 A/G was significantly associated with DC treatment response. The presence of a G allele indicated poor response to the therapeutic vaccine (p=0.0031; OR=33.00; CI=1.74-624.66), and the SNP behaved in a dominant model (A/A vs. A/G+G/G p=0.0009; OR=107.66; 95% CI=3.85-3013.31), being the A/G genotype present only in weak/transient responders, conferring susceptibility to poor response to the immune treatment. DISCUSSION: CNOT1 is known to be involved in the control of mRNA deadenylation and mRNA decay. Moreover, CNOT1 has been recently described as being involved in the regulation of inflammatory processes mediated by tristetraprolin (TTP). The TTP-CCR4-NOT complex (CNOT1 in the CCR4-NOT complex is the binding site for TTP) has been reported as interfering with HIV replication, through post-transcriptional control. Therefore, we can hypothesize that genetic variation occurring in the CNOT1 gene could impair the TTP-CCR4-NOT complex, thus interfering with HIV replication and/or host immune response. CONCLUSIONS: Being aware that our findings are exclusive to the 18 patients studied with a need for replication, and that the genetic variant of CNOT1 gene, localized at intron 3, has no known functional effect, we propose a novel potential candidate locus for the modulation of the response to the immune treatment, and open a discussion on the necessity to consider the host genome as another potential variant to be evaluated when designing an immune therapy study

Constitutive gene expression profile segregates toxicity in locally advanced breast cancer patients treated with high-dose hyperfractionated radical radiotherapy

[EN] Breast cancer patients show a wide variation in normal tissue reactions after radiotherapy. The individual sensitivity to x-rays limits the efficiency of the therapy. Prediction of individual sensitivity to radiotherapy could help to select the radiation protocol and to improve treatment results. The aim of this study was to assess the relationship between gene expression profiles of ex vivo un-irradiated and irradiated lymphocytes and the development of toxicity due to high-dose hyperfractionated radiotherapy in patients with locally advanced breast cancer. Raw data from microarray experiments were uploaded to the Gene Expression Omnibus Database http://www.ncbi.nlm.nih.gov/geo/ (GEO accession GSE15341). We obtained a small group of 81 genes significantly regulated by radiotherapy, lumped in 50 relevant pathways. Using ANOVA and t-test statistical tools we found 20 and 26 constitutive genes (0 Gy) that segregate patients with and without acute and late toxicity, respectively. Non-supervised hierarchical clustering was used for the visualization of results. Six and 9 pathways were significantly regulated respectively. Concerning to irradiated lymphocytes (2 Gy), we founded 29 genes that separate patients with acute toxicity and without it. Those genes were gathered in 4 significant pathways. We could not identify a set of genes that segregates patients with and without late toxicity. In conclusion, we have found an association between the constitutive gene expression profile of peripheral blood lymphocytes and the development of acute and late toxicity in consecutive, unselected patients. These observations suggest the possibility of predicting normal tissue response to irradiation in high-dose non-conventional radiation therapy regimens. Prospective studies with higher number of patients are needed to validate these preliminary results.

Myc-mediated control of gene transcription in cancer cells

The Myc oncoproteins belong to a family of transcription factors composed by Myc, N-Myc and L-Myc. The most studied components of this family are Myc and N-Myc because their expressions are frequently deregulated in a wide range of cancers. These oncoproteins can act both as activators or repressors of gene transcription. As activators, they heterodimerize with Max (Myc associated X-factor) and the heterodimer recognizes and binds a specific sequence elements (E-Box) onto gene promoters recruiting histone acetylase and inducing transcriptional activation. Myc-mediated transcriptional repression is a quite debated issue. One of the first mechanisms defined for the Myc-mediated transcriptional repression consisted in the interaction of Myc-Max complex Sp1 and/or Miz1 transcription factors already bound to gene promoters. This interaction may interfere with their activation functions by recruiting co-repressors such as Dnmt3 or HDACs. Moreover, in the absence of , Myc may interfere with the Sp1 activation function by direct interaction and subsequent recruitment of HDACs. More recently the Myc/Max complex was also shown to mediate transcriptional repression by direct binding to peculiar E-box. In this study we analyzed the role of Myc overexpression in Osteosarcoma and Neuroblastoma oncogenesis and the mechanisms underling to Myc function. Myc overexpression is known to correlate with chemoresistance in Osteosarcoma cells. We extended this study by demonstrating that c-Myc induces transcription of a panel of ABC drug transporter genes. ABCs are a large family trans-membrane transporter deeply involved in multi drug resistance. Furthermore expression levels of Myc, ABCC1, ABCC4 and ABCF1 were proved to be important prognostic tool to predict conventional therapy failure. N-Myc amplification/overexpression is the most important prognostic factor for Neuroblastoma. Cyclin G2 and Clusterin are two genes often down regulated in neuroblastoma cells. Cyclin G2 is an atypical member of Cyclin family and its expression is associated with terminal differentiation and apoptosis. Moreover it blocks cell cycle progression and induces cell growth arrest. Instead, CLU is a multifunctional protein involved in many physiological and pathological processes. Several lines of evidences support the view that CLU may act as a tumour suppressor in Neuroblastoma. In this thesis I showed that N-Myc represses CCNG2 and CLU transcription by different mechanisms. • N-Myc represses CCNG2 transcription by directly interacting with Sp1 bound in CCNG2 promoter and recruiting HDAC2. Importantly, reactivation of CCNG2 expression through epigenetic drugs partially reduces N-Myc and HDAC2 mediated cell proliferation. • N-Myc/Max complex represses CLU expression by direct binding to a peculiar E-box element on CLU promoter and by recruitment of HDACs and Polycomb Complexes, to the CLU promoter. Overall our findings strongly support the model in which Myc overexpression/amplification may contribute to some aspects of oncogenesis by a dual action: i) transcription activation of genes that confer a multidrug resistant phenotype to cancer cells; ii), transcription repression of genes involved in cell cycle inhibition and cellular differentiation.

New approach in the diagnosis and therapy of hyperphenylalaninemia

Background. Phenylketonuria is the most prevalent inborn error of aminoacid metabolism. Is an autosomal recessive disorder. It results from mutations in the phenylalanine hydroxilase (PAH) gene. Phenotypes can vary from mild hyperphenylalaninemia to a severe phenylketonuria wich, if untreated, results in severe mental retardation. Thanks to neonatal screening programmes, early detection and promp dietetic intervention (phenylalanine restricted diet lifelong) has allowed to avoid neurocognitive complications. Recently, a new therapy is become widely used: the oral supplementation with the PAH cofactor (BH4), wich can alleviate the diet burden. Genotype-phenotype correlation is a reliable tool to predict metabolic phenotype in order to establish a better tailored diet and to assess the potential responsiveness to BH4 therapy. Aim Molecular analysis of the PAH gene, evaluation of genotype-phenotype correlation and prediction of BH4 responsiveness in a group of HPA patients living in Emilia Romagna. Patients and methods. We studied 48 patients affected by PAH deficiency in regular follow-up to our Metabolic Centre. We performed the molecular analysis of these patients using genomic DNA extracted from peripheral blood samples Results. We obtained a full genotipic characterization of 46 patients. We found 87 mutant alleles and 35 different mutations, being the most frequent IVS10-11 G>A (19.3%), R261Q (9.1%), R158Q (9.1%), R408Q (6.8%) and A403V (5.7%), including 2 new ones (L287, N223Y) ever described previously. Notably, we found 15 mutations already identified in BH4-responsive patients, according to the literature. We found 42 different genotipic combinations, most of them in single patients and involving a BH4-responsive mutation. Conclusion. BH4 responsiveness is shown by a consistent number of PAH deficient hyperphenylalaninemic patients. This treatment, combined with a less restricted diet or as monotherapy, can reduce nutritional complications and improve the quality of life of these patients.

Targeting the p53–MDM2 interaction by the small-molecule MDM2 antagonist Nutlin-3a: a new challenged target therapy in adult Philadelphia positive acute lymphoblastic leukemia patients

The human p53 tumor suppressor, known as the “guardian of the genome”, is one of the most important molecules in human cancers. One mechanism for suppressing p53 uses its negative regulator, MDM2, which modulates p53 by binding directly to and decreasing p53 stability. In testing novel therapeutic approaches activating p53, we investigated the preclinical activity of the MDM2 antagonist, Nutlin-3a, in Philadelphia positive (Ph+) and negative (Ph-) leukemic cell line models, and primary B-Acute lymphoblastic leukemia (ALL) patient samples. In this study we demonstrated that treatment with Nutlin-3a induced grow arrest and apoptosis mediated by p53 pathway in ALL cells with wild-type p53, in time and dose-dependent manner. Consequently, MDM2 inhibitor caused an increase of pro-apoptotic proteins and key regulators of cell cycle arrest. The dose-dependent reduction in cell viability was confirmed in primary blast cells from Ph+ ALL patients with the T315I Bcr-Abl kinase domain mutation. In order to better elucidate the implications of p53 activation and to identify biomarkers of clinical activity, gene expression profiling analysis in sensitive cell lines was performed. A total of 621 genes were differentially expressed (p < 0.05). We found a strong down-regulation of GAS41 (growth-arrest specific 1 gene) and BMI1 (a polycomb ring-finger oncogene) (fold-change -1.35 and -1.11, respectively; p-value 0.02 and 0.03, respectively) after in vitro treatment as compared to control cells. Both genes are repressors of INK4/ARF and p21. Given the importance of BMI in the control of apoptosis, we investigated its pattern in treated and untreated cells, confirming a marked decrease after exposure to MDM2 inhibitor in ALL cells. Noteworthy, the BMI-1 levels remained constant in resistant cells. Therefore, BMI-1 may be used as a biomarker of response. Our findings provide a strong rational for further clinical investigation of Nutlin-3a in Ph+ and Ph-ALL.

Molecular mechanisms of shikonin and its derivatives in cancer therapy

The identification of molecular processes involved in cancer development and prognosis opened avenues for targeted therapies, which made treatment more tumor-specific and less toxic than conventional therapies. One important example is the epidermal growth factor receptor (EGFR) and EGFR-specific inhibitors (i.e. erlotinib). However, challenges such as drug resistance still remain in targeted therapies. Therefore, novel candidate compounds and new strategies are needed for improvement of therapy efficacy. Shikonin and its derivatives are cytotoxic constituents in traditional Chinese herbal medicine Zicao (Lithospermum erythrorhizin). In this study, we investigated the molecular mechanisms underlying the anti-cancer effects of shikonin and its derivatives in glioblastoma cells and leukemia cells. Most of shikonin derivatives showed strong cytotoxicity towards erlotinib-resistant glioblastoma cells, especially U87MG.ΔEGFR cells which overexpressed a deletion-activated EGFR (ΔEGFR). Moreover, shikonin and some derivatives worked synergistically with erlotinib in killing EGFR-overexpressing cells. Combination treatment with shikonin and erlotinib overcame the drug resistance of these cells to erlotinib. Western blotting analysis revealed that shikonin inhibited ΔEGFR phosphorylation and led to corresponding decreases in phosphorylation of EGFR downstream molecules. By means of Loewe additivity and Bliss independence drug interaction models, we found erlotinb and shikonin or its derivatives corporately suppressed ΔEGFR phosphorylation. We believed this to be a main mechanism responsible for their synergism in U87MG.ΔEGFR cells. In leukemia cells, which did not express EGFR, shikonin and its derivatives exhibited even greater cytotoxicity, suggesting the existence of other mechanisms. Microarray-based gene expression analysis uncovered the transcription factor c-MYC as the commonly deregulated molecule by shikonin and its derivatives. As validated by Western blotting analysis, DNA-binding assays and molecular docking, shikonin and its derivatives bound and inhibited c-MYC. Furthermore, the deregulation of ERK, JNK MAPK and AKT activity was closely associated with the reduction of c-MYC, indicating the involvement of these signaling molecules in shikonin-triggered c-MYC inactivation. In conclusion, the inhibition of EGFR signaling, synergism with erlotinib and targeting of c-MYC illustrate the multi-targeted feature of natural naphthoquinones such as shikonin and derivatives. This may open attractive possibilities for their use in a molecular targeted cancer therapy.