Chemotherapeutic Drug Resistance Research Articles

The significance of heat shock proteins in breast cancer therapy

The evalutionary conserved heat shock proteins are involved basically life protecting mechanisms against harmful extracellular effects such as primarily heat shock response. Normally, the expression of these proteins is increased for cellular adaptation to high temperature. This increase is also important in the etiology of breast cancer. Overexpression of heat shock proteins is associated with reduced disease-free survival in breast cancer. However, increased expression of these proteins is related to acquired resistance of traditional chemotherapeutic drugs in use in breast cancer treatment. In this review, we discuss the multiple roles of heatshock proteins in resistance and where we are to overcome this in clinical practice.

MiRNA-200c increases the sensitivity of breast cancer cells to doxorubicin through the suppression of E-cadherin-mediated PTEN/Akt signaling

Doxorubicin (ADR) is successfully used to treat breast cancer, however, it is often associated with the acquired resistance of breast cancer cells which eliminates the therapeutic efficiency of ADR, leading to relapse and a poorer prognosis. It has been reported that microRNA-200c (miRNA-200c), a non-coding RNA, is important in the epithelial to mesenchymal transition (EMT) and metastasis in breast cancer cells. Recent evidence demonstrated that miRNA-200c is also regulated in chemotherapeutic drug resistance, however, the precise mechanism by which this occurs remains unclear. In this study, we demonstrated that the loss of miRNA-200c correlates with the acquired resistance of breast cancer cells to ADR. In addition, the loss of miRNA-200c correlated with decreased levels of E-cadherin and PTEN, and increased levels of ZEB1 and phospho-Akt (p-Akt) in ADR-resistant breast cancer cells (MCF-7/ADR cells). More importantly, we demonstrated that the gain of miRNA-200c results in an increased sensitivity of cells to ADR, downregulation of ZEB1, upregulation of E-cadherin and PTEN, and inactivation of Akt signaling. Following the co-transfection of E-cadherin siRNA, the miRNA-200c-mediated regulation of Akt signaling and PTEN was inhibited. Results of the present study also demonstrated that Akt signaling is involved in the ADR resistance of breast cancer cells since LY294002, an inhibitor of Akt signaling, partially restored the sensitivity of MCF-7/ADR cells to ADR. In conclusion, miRNA-200c inhibited Akt signaling through its effects on E-cadherin and PTEN, resulting in the inhibition of ADR resistance in breast cancer cells.

High Expression of Lewis y Antigen and CD44 Is Correlated with Resistance to Chemotherapy in Epithelial Ovarian Cancers

ObjectivesTo measure Lewis y antigen and CD44 antigen expression in epithelial ovarian carcinoma and to correlate the levels of these antigens with clinical response to chemotherapy.MethodsThe study cases included 34 cases of ovarian carcinoma with resistance to chemotherapeutic drugs, 6 partially drug-sensitive cases, and 52 drug-sensitive cases (92 total).ResultsThe rates of expression of Lewis y antigen and CD44 antigen were significantly greater in the drug-resistant group than that in the partially-sensitive or sensitive groups. Surgical stage, residual tumor size and expression of CD44 and Lewis y antigen in ovarian carcinoma tissues were independent risk factors for chemotherapeutic drug resistance.ConclusionsOver-expression of Lewis y and CD44 antigen are strong risk factors for chemotherapeutic drug resistance in ovarian carcinoma patients.

Combination Effect of Paclitaxel and Hyaluronic Acid on Cancer Stem-Like Side Population Cells

Cancer recurrence is the main cause of chemotherapeutic treatment failure. The mechanisms driving cancer recurrence may be due to very rare subpopulation cells, cancer stem-like cells (CSCs). Therefore, the early detection and better treatment of cancer stem-like cells are of great interest. In this study, we investigated how to eliminate the side population cells (SP), which have the characteristics of cancer stem-like cells, and also show chemotherapy resistance. Fluorescence-activated cell sorting (FACS) were used to sort SP and non-SP cells from human liver cancers, Huh-7 Hyaluronic acid (HA), which is an abundant component in the extracellular matrix, is known to involve in proliferation of normal and cancer cells. Herein, we investigated the effect of HA component on chemotherapy against SP cells. Cell growth inhibitory effects of the paclitaxel (PTX) chemotherapy combined with the HA component on SP cells of Huh-7 was determined using the trypan blue dye exclusion test. PTX combined with HA was found to show more increased inhibition of cell growth in both SP and non-SP cells, compared to free PTX treatment. In conclusion, SP cells of Huh-7 shows chemotherapeutic drug resistance due to the over-expressed efflux pumps. HA proposed one of possibilities to overcome the limitation of chemotherapy against cancer stem-like cells.

Long interspersed nuclear element ORF-1 protein promotes proliferation and resistance to chemotherapy in hepatocellular carcinoma

To clarify the specific roles and mechanisms of long interspersed nuclear element-1 ORF-1 protein [human long interspersed nuclear element-1 (LINE-1), ORF-1p] in chemotherapeutic drug resistance and cell proliferation regulation in hepatocellular carcinoma (HCC) cells. MTT assays were performed to identify the effect of the chemotherapeutic drug toxicity on HepG2 cells. Cell proliferation inhibition and the IC50 were calculated by the Origin 8.0 software. Western blotting assays were performed to investigate whether LINE-1 ORF-1p modulates the expression of some important genes, including p53, p27, p15, Bcl-2, mdr, and p-gp. To corroborate the proliferation and anchor-independent growth results, the HepG2 cells were analyzed by flow cytometry to investigate the effect of LINE-1 ORF-1p on the apoptosis regulation. LINE-1 ORF-1p contributed to the resistance to several chemotherapeutic drugs (cisplatin and epirubicin) in HepG2 cells. The IC50 of the epirubicin and cisplatin increased from 36.04 nmol/L to 59.11 nmol/L or from 37.94 nmol/L to 119.32 nmol/L. Repression of LINE-1 ORF-1p expression by the siRNA could markedly enhance the response of HepG2 cells to the epirubicin and cisplatin. The IC50 correspondingly decreased from 28.06 nmol/L to 3.83 nmol/L or from 32.04 nmol/L to 2.89 nmol/L. Interestingly, down-regulation of LINE-1 ORF-1p level by siRNA could promote the response of HepG2 cells to the paclitaxel. The IC50 decreased from 35.90 nmol/L to 7.36 nmol/L. However, overexpression of LINE-1 ORF-1p did not modulate the paclitaxel toxicity in HepG2 cells. Further Western blotting revealed that LINE-1 ORF-1p enhanced mdr and p-gp gene expression. As a protein arrested in the nucleus, LINE-1 ORF-1p may function through modulating transcriptional activity of some important transcription factors. Indeed, LINE-1 ORF-1p promoted HepG2 cell proliferation, anchor-independent growth and protected the cells against apoptosis through modulating the expression of p15, p21, p53, and Bcl-2 genes. LINE-1 ORF-1p promotes HepG2 cell proliferation and plays an important role in the resistance of chemotherapeutic drugs. By establishing novel roles and defining the mechanisms of LINE-1 ORF-1p in HCC chemotherapeutic drug resistance and cell proliferation regulation, this study indicates that LINE-1 ORF-1p is a potential target for overcoming HCC chemotherapeutic resistance.

Editorial on research topic: aldo-keto reductases and role in human disease

Research on Aldo-keto reductases (AKRs) over the years has revealed a remarkable diversity in the reactivity of these soluble NAD(P)(H) oxidoreductases. The AKR enzymes primarily catalyze the reduction of aldehydes and ketones to primary and secondary alcohols, respectively. The 10 known human AKR enzymes can turnover a vast range of endogenous and exogenous substrates, including glucose, steroids, carcinogens, reactive aldehydes, and a variety of carbonyl-containing drugs. These enzymes have been implicated in a number of human diseases, including cancer and disorders of endocrinology and metabolism. AKRs are involved in the development and progression of many cancers, as well as chemotherapeutic drug resistance. AKR1B1 and AKR1B10 are overexpressed in tumors, such as liver, breast, and lung cancer. Several AKRs (AKR1A1, AKR1B10, and AKR1C1-3) are involved in tobacco-carcinogenesis, but they also catalyze the detoxication of nicotine derived nitrosamino ketones. In addition, AKR1C1-3 enzymes play a key role in the regulation of proliferative signaling in hormone dependent cancers. AKR1B1 has long been known for its involvement in diabetic complications. Recent studies now suggest AKR1B enzymes play a key role in inflammatory diseases such as atherosclerosis, sepsis, asthma, uveitis, and colon cancer by mediating oxidative stress-induced inflammatory signals. In recognition of the role of AKR in various diseases, significant efforts have been made in the development of specific/selective AKR1B1 and AKR1C3 inhibitors. The aim of this Research Topic forum is to celebrate the advancements seen in the AKR field via review papers and original articles, as well as to promote future research in understanding the underlying mechanism(s) of the role of AKRs in human disease. Eight review articles are included in this issue, and are grouped into disease areas (cancer, disorders of endrocrinology and metabolism) after the artile written by Chen and Zhang (2012) which describes the current understanding of the regulation of all human AKR members in relation to disease. The significance of the AKR enzymes for research in various cancers is discussed in four review articles. The article by Matsunaga et al. (2012) focuses on the role AKR1B10 in drug resistance of cancer cells and the development of AKR1B10 inhibitors to reverse chemoresistance. The article by Ruiz et al. (2012) describes the retinaldehyde reductase activity of AKRs in relation to proliferation and tumorigenesis. The paper from the Penning group (Zhang et al., 2012) summarizes the role of AKR in the metabolic activation and detoxication of polycyclic aromatic hydrocarbons and the relevance of phase II conjugation reactions to human lung carcinogenesis. Rižner (2012) provides a review on the role played by AKR1B and AKR1C members in the pathogenesis of endometrial and cervical cancers, as well as other uterine diseases. The aspect that members of AKRs can mediate the timing of parturition through metabolism of progesterone and prostaglandins is discussed by Byrns (2012). The role of AKR1B members in modification and production of signaling molecules is emphasized by Pastel et al. (2012) for a better understanding of the physiological and pathological role of AKR1B enzymes. The article by Tang et al. (2012) focuses on the increases in oxidative stress by AKR1B1 in pathogenesis of diabetic complications such as cardiovascular diseases. Three Original Research Articles follow the review articles. The article by Fortier and coworkers (Bresson et al., 2012) reports on the prostaglandin F synthase activity of AKR1B1 in different models of living cells and tissues. This activity of AKR1B1 represents a novel target to regulate ischemic and inflammatory responses associated with several human pathologic conditions. The article by Laffin and Petrash (2012) examines the expression of AKR1B1 and AKR1B10 across all major human cancer types. Their findings point to the great potential of AKR1B1 inhibition as novel cancer therapeutics. Schulze et al. (2012) describe their studies on basal and regulatory promoter of AKR1C3 in relation to prostate cancer. As pointed out in the review article by Pastel et al. (2012) there is a “marked contrast between the abundance of enzymatic data and the small number of reports dedicated to functional studies in a physiological context.” It is my hope that with an emphasis on the role of AKRs in human disease the articles included in this issue will benefit a broad audience and the researchers in the rapidly growing AKR filed.

Plk1 Phosphorylation of Orc2 and Hbo1 Contributes to Gemcitabine Resistance in Pancreatic Cancer

Although gemcitabine is the standard chemotherapeutic drug for treatment of pancreatic cancer, almost all patients eventually develop resistance to this agent. Previous studies identified Polo-like kinase 1 (Plk1) as the mediator of gemcitabine resistance, but the molecular mechanism remains unknown. In this study, we show that Plk1 phosphorylation of Orc2 and Hbo1 mediates the resistance to gemcitabine. We show that the level of Plk1 expression positively correlates with gemcitabine resistance, both in pancreatic cancer cells and xenograft tumors. Overexpression of Plk1 increases gemcitabine resistance, while inhibition of Plk1 sensitizes pancreatic cancer cells to gemcitabine treatment. To validate our findings, we show that inhibition of Plk1 sensitizes tumors to gemcitabine treatment in a mouse xenograft study. Mechanistically, we find that Plk1 phosphorylation of Orc2 maintains DNA replication on gemcitabine treatment. Furthermore, Plk1 phosphorylation of Hbo1 transcriptionally increases cFos expression and consequently elevates its target multidrug resistance 1 (MDR1), which was previously reported to confer chemotherapeutic drug resistance. Knockdown of cFos or MDR1 sensitizes gemcitabine-resistant cells to gemcitabine treatment. Finally, pancreatic cancer cells expressing Plk1-unphosphorylatable mutants of Orc2 or Hbo1 are more sensitive to gemcitabine than cells expressing wild-type Orc2 or Hbo1. In short, our study provides a mechanism for Plk1-mediated gemcitabine resistance, suggesting that Plk1 is a promising target for treatment of gemcitabine-resistant pancreatic cancer.

Elevated Levels of Lewis Y and Integrin α5β1 Correlate with Chemotherapeutic Drug Resistance in Epithelial Ovarian Carcinoma

ObjectiveTo measure Lewis y and integrin α5β1 expression in epithelial ovarian carcinoma and to correlate the levels of these molecules with ovarian carcinoma chemotherapy and prognosis.MethodsThe study population included 34 ovarian carcinoma patients with chemotherapeutic drug-resistance, six partially drug-sensitive cases, and 52 drug-sensitive cases (92 total). Immunochemistry was used to determine expression of Lewis y antigen and integrin α5β1 in ovarian carcinoma tissues, and correlation of these molecules with chemotherapy resistance was further investigated, Multi-factor logistic regression analysis was applied to investigate: age, surgical stage, grade, subtype of patient cases, metastasis of lymph nodes, residual tumor size, expression levels of Lewis y antigen and integrin α5β1 correlation with ovarian carcinoma chemotherapy resistance.ResultsThe expression rates of Lewis y antigen and integrins α5 and β1 were significantly greater in the drug-resistant group (91.17%, 85.29%, 88.24%) than the partially sensitive (50.00%, 33.33%, 50.00%) or sensitive groups (61.54%, 57.69%, 55.77%). Binary logistic regression analysis revealed that surgical stage, residual tumor size, and expression of integrin α5 and Lewis y in ovarian carcinoma tissues were independent risk factors for chemotherapeutic drug resistance.ConclusionsOverexpression of Lewis y and integrin α5 are strong risk factors for chemotherapeutic drug resistance in ovarian carcinoma patients.

Role of the forkhead transcription factor FOXO-FOXM1 axis in cancer and drug resistance

The forkhead transcription factors FOXO and FOXM1 have pivotal roles in tumorigenesis and in mediating chemotherapy sensitivity and resistance. Recent research shows that the forkhead transcription factor FOXM1 is a direct transcriptional target repressed by the forkhead protein FOXO3a, a vital downstream effector of the PI3K-AKT-FOXO signaling pathway. Intriguingly, FOXM1 and FOXO3a also compete for binding to the same gene targets, which have a role in chemotherapeutic drug action and sensitivity. An understanding of the role and regulation of the FOXO-FOXM1 axis will impact directly on our knowledge of chemotherapeutic drug action and resistance in patients, and provide new insights into the design of novel therapeutic strategy and reliable biomarkers for prediction of drug sensitivity.

Effects of Ectopic Expression of NGAL on Doxorubicin Sensitivity

Neutrophil gelatinase-associated lipocalin (NGAL, a.k.a Lnc2) is a member of the lipocalin family which has diverse roles including stabilizing matrix metalloproteinase-9 from auto-degradation and as siderocalins which are important in the transport of iron. NGAL also has important biological functions involved in immunity and inflammation as well as responses to kidney damage. NGAL expression has also been associated with certain neoplasia and is important in the metastasis of breast cancer. Many advanced cancer patients have elevated levels of NGAL in their urine and it has been proposed that NGAL may be a prognostic indicator for certain cancers (e.g. breast, brain, and others). NGAL expression is detected in response to various chemotherapeutic drugs including doxorubicin and docetaxel. We were interested in the roles of NGAL expression in cancer and whether it is associated with chemotherapeutic drug resistance. In the present study, we investigated whether increased NGAL expression led to resistance to the chemotherapeutic drug doxorubicin in normal breast epithelial cells (MCF-10A), breast cancer cells (MCF-7), and colorectal cancer cells (HT-29). We infected the various cell lines with a retrovirus encoding NGAL which we constructed. Increased NGAL expression was readily detected in the NGAL-infected cells but not the empty vector-infected cells. However, increased NGAL expression did not alter the sensitivity of the cells to the chemotherapeutic drug doxorubicin. Thus, although NGAL expression is often detected after chemotherapeutic drug treatment, it by itself, does not lead to doxorubicin resistance.

RNA interference screening identifies a novel role for autocrine fibroblast growth factor signaling in neuroblastoma chemoresistance

Chemotherapeutic drug resistance is one of the major causes for treatment failure in high-risk neuroblastoma (NB), the most common extra cranial solid tumor in children. Poor prognosis is typically associated with MYCN amplification. Here, we utilized a loss-of-function kinome-wide RNA interference screen to identify genes that cause cisplatin sensitization. We identified fibroblast growth factor receptor 2 (FGFR2) as an important determinant of cisplatin resistance. Pharmacological inhibition of FGFR2 confirmed the importance of this kinase in NB chemoresistance. Silencing of FGFR2 sensitized NB cells to cisplatin-induced apoptosis, which was regulated by the downregulation of the anti-apoptotic proteins BCL2 and BCLXL. Mechanistically, FGFR2 was shown to activate protein kinase C-δ to induce BCL2 expression. FGFR2, as well as the ligand fibroblast growth factor-2, were consistently expressed in primary NB and NB cell lines, indicating the presence of an autocrine loop. Expression analysis revealed that FGFR2 correlates with MYCN amplification and with advanced stage disease, demonstrating the clinical relevance of FGFR2 in NB. These findings suggest a novel role for FGFR2 in chemoresistance and provide a rational to combine pharmacological inhibitors against FGFR2 with chemotherapeutic agents for the treatment of NB.

Mutations and Deregulation of Ras/Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR Cascades Which Alter Therapy Response.

The Ras/Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR cascades are often activated by genetic alterations in upstream signaling molecules such as receptor tyrosine kinases (RTK). Certain components of these pathways, RAS, NF1, BRAF, MEK1, DUSP5, PP2A, PIK3CA, PIK3R1, PIK3R4, PIK3R5, IRS4, AKT, NFKB1, MTOR, PTEN, TSC1, and TSC2 may also be activated/inactivated by mutations or epigenetic silencing. Upstream mutations in one signaling pathway or even in downstream components of the same pathway can alter the sensitivity of the cells to certain small molecule inhibitors. These pathways have profound effects on proliferative, apoptotic and differentiation pathways. Dysregulation of components of these cascades can contribute to: resistance to other pathway inhibitors, chemotherapeutic drug resistance, premature aging as well as other diseases. This review will first describe these pathways and discuss how genetic mutations and epigenetic alterations can result in resistance to various inhibitors.

Transcriptional regulation of MDR-1 by HOXC6 in multidrug-resistant cells

Resistance to chemotherapeutic drugs is a significant clinical problem in the treatment of cancer and this resistance has been linked to the cellular expression of multidrug-efflux transporters. The aim of this study was to explore the role of HOXC6 in the regulation of multidrug resistance (MDR) to chemotherapeutic drugs. The HOXC6 gene was identified as being overexpressed in drug-resistant cells compared with parental cell lines. Transfection assays demonstrated that HOXC6 activated MDR-1 promoter activity. A series of MDR-1 promoter deletion mutants was examined and the minimal HOXC6-responsive region was identified to be in the TAAT motif (-2243 bp) of the MDR-1 promoter. Interestingly, overexpression of HOXC6 in the parental cell lines resulted in the upregulation of MDR-1 expression. The inhibition of HOXC6 using small interfering RNA led to the repression of MDR-1. We determined that knockdown of HOXC6 expression in MDR cells increased their sensitivity to paclitaxel. Flow cytometry analysis suggested that siHOXC6 could induce paclitaxel-induced apoptosis and that this was accompanied by an increased accumulation and a decreased release of paclitaxel. Taken together, our findings suggest that HOXC6 expression is an important mechanism of chemotherapeutic drug resistance via its regulation of MDR-1.

Rapid culture diagnosis of tuberculous lymphadenitis from a tertiary care centre in an endemic nation: Potential and pitfalls

In spite of low sensitivity and specificity, standard diagnostic algorithm recommends fine needle aspiration cytology (FNAC) and direct microscopic screening for acid-fast bacilli (AFB) for the routine diagnosis of tuberculous lymphadenopathy (LNTB). In this study, the diagnostic utility of liquid broth based automated culture (BacT/ALERT 3D) technique was assessed in comparison with conventional techniques in 89 clinically suspected tubercular lymphadenitis patients. 60% (n = 53) were positive by FNAC and 38.4% (n = 34) demonstrated AFB in smear examination. BacT/ALERT yielded isolation in 43.1% (n = 38) aspirates, confirming tubercular aetiology. We also found six paediatric culture-positive cases which showed negative outcome by both FNAC and smear. Thus, we conclude that culture by BacT/ALERT, may be used for faster yield of Mycobacteria in LNTB, especially in children. Additionally, this could also be used as a platform for further differentiation of Mycobacterium tuberculosis from non-tuberculous mycobacteria (NTM) infection and for testing of anti-tubercular chemotherapeutic agents whenever drug resistance is suspected

The role and significance of FoxM1 in invasive breast cancer.

1056 Background: The Forkhead Box protein M1 (FoxM1) is known to regulate a variety of biologic processes in mammalian cells including cell growth and survival, angiogenesis, DNA damage response, chemotherapeutic drug resistance, and cancer cell migration and invasion. We evaluate the role and significance of Fox M1 in primary breast cancer in vitro and analyzed the relation with FoxM1 expression and clinicopathologic features. Methods: Immunohistochemical staining was used for evaluation of cytoplasmic expression of FoxM1 with TMA of invasive breast cancer. In various breast cancer cell lines, we evaluated FoxM1 expression and treated docetaxel/cisplatin in combination with Siomycin A (FoxM1 inhibitor) for BT20 cell line. Results: From Nov 1995 to Jul 2007, in 84 patients with stage 1-3 invasive breast cancer, FoxM1 expression was noted in 58.7%. Median follow-up duration was 85.1 months. Lymphovascular invasion was positively correlated with FoxM1 expression (p=0.040). In multivariate analysis, FoxM1 expression (p=0.005), HR negativity (p=0.002), high histologic grade (p=0.023), hign nuclear grade (p=0.045), lymphovascular invasiveness (p=0.017), and stage 3 cancer (p=0.015) matched poor disease-free survival. In vitro study, FoxM1 was expressed BT474, JIMT-1, BT20, HCC-1937, and MDA-MB-231 cell lines. The inhibition of FoxM1 had synergistic effect on cisplatin treatment, not docetaxel in BT20 cell. Conclusions: FoxM1 expression was noted in triple negative breast cancer cell lines and its inhibition had synergistically cytotoxic effect on BT20 cell line in combination with cisplatin. Although the further in vivo and clinical study should be needed to draw the solid conclusions, FoxM1 could be both a promising target of treatment for triple negative breast cancer and a independent prognostic factor.

Prostate cancer inhibitory activity of a novel dual inhibitor, EL102, in combination with docetaxel, and its effects on MDR1-mediated drug resistance in vitro.

e15126 Background: EL102 is a dual-action drug promoting apoptosis and inhibiting angiogenesis. It exerts its action though the inhibition of Hif1a induced hypoxic signalling and induction of the Caspase 3/7 apoptotic cascade. The drug has equal activity in normoxia and hypoxia indicating it may be equally active in these different tumor compartments. We tested its ability to circumvent chemotherapeutic drug resistance. Methods: We assessed the ability of EL102 to inhibit prostate cancer cell proliferation and motility in vitro, calculating IC50s for CWR22, 22Rv2, PC3 and DU145 prostate cancer cell lines, comparing sensitivity between androgen dependent, androgen independent and metastatic prostate cancer. Additionally we assessed the activity of EL102 in combination with docetaxel in vitro and in murine CWR22 xenografts. The ability to overcome MDR1 and BCRP mediated drug resistance was also tested using DLKP drug resistant variants which exhibit 200 fold resistance to doxorubicin, docetaxel, paclitaxel and vincristine. Results: We found that prostate cancer cell lines are sensitive to EL102 with IC50s in the region of 10-50nM. Of particular interest was the identical sensitivity of the androgen independent 22Rv1 and its androgen dependent parent CWR22, suggesting ability to overcome hormone refractory prostate cancer. Additionally we demonstrate dose response for inhibition of cell motility in metastatic DU145. In CWR22 murine mouse models treatment with EL102 resulted in decreased tumor volume compared to control. A docetaxel and EL102 combination arm demonstrated the greater inhibition of tumor growth than EL102 or docetaxel alone. The lung cancer cell line DLKP, its drug resistant variants DLKPA (MDR1 overexpressing) and DLKPMitox (BCRP overexpressing) were equally sensitive to EL102 indicating that EL102 is not a substrate for MDR1 or BCRP. Conclusions: EL102 is a potential therapeutic for the treatment of prostate cancer, in particular in combination with docetaxel, and exhbibits the potential to overcome drug resistane. Future studies will include the efficacy of this drug in prostate cancer metastatic mouse models.

Abstract 920: Effective elimination of drug resistsant melanoma cells by α-phenylethyl isothiocyanate (PEITC)

Abstract Drug resistance is a major problem in clinical treatment of malignant melanoma. A characteristic feature of melanoma is the abnormally high levels of basal free radicals produced in transformed melanosomes when compared to normal melanocytes. Reactive oxygen species (ROS) activate pro-oncogenic signaling pathways that promote cancer progression, metastasis and contribute to chemotherapeutic drug resistance. However, an excessive overload of ROS in cells beyond a certain tolerance limit may trigger oxidative damage and cell death. Thus cellular ROS modulation presents a valuable therapeutic strategy for melanoma. Based on the above observations, we hypothesize that causing ROS agglomeration in cells, either through depletion of antioxidants or by initiating uncontrolled ROS production, is a plausible strategy to selectively kill melanoma cells, and that α-phenylethyl isothiocyanate (PEITC), a natural compound known to disable cellular GSH antioxidant system, will be effective for this purpose. We showed that that PEITC exhibited potent inhibitory effect on malignant melanoma cells with an average IC50 value of approximately 3 µM. Interestingly, MEL624 and HS294T cells, which are resistant to existing antitumor agents used in melanoma treatment such as AZD6244 and Vemurafenib, were highly sensitive to PEITC. The cell death was primarily apoptotic; the effect of PEITC could be partially reversed using the pan-caspase inhibitor Z-VAD. Furthermore, treatment of cells with PEITC lead to an aberrant increase in reactive oxygen species levels, as evidenced by FACS analysis using the fluorescent ROS dyes DCF-DA, DAF-FM and dihyroethidium. ROS increase upon PEITC treatment occurred as early as one hour, indicating that seemed to a primary event leading to subsequent cell damage. This increase in ROS was associated with a decrease in the glutathione levels in cells, suggesting that depletion of the glutathione antioxidant could be the reason for the abnormal ROS elevation induced by PEITC. This was further verified by using PEITC in combination with the antioxidant N-acetyl cysteine, which completely rescued the cells from the toxic effect of PEITC. Importantly, normal melanocytes were minimally sensitive to PEITC. In addition, combination of PEITC with other standard melanoma drugs like carmustine and temozolomide exhibited synergistic effect, suggesting the possibility of using this compound to overcome drug resistance. This was further validated through in vivo studies in mice, where a combination of PEITC with BCNU seemed to reduce the tumor burden. The cytotoxic effect of PEITC was also validated in primary cancer cells isolated from melanoma specimens from patients. Thus PEITC proves to be an effective antimelanoma agent that warrants further investigation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 920. doi:1538-7445.AM2012-920

Abstract 1892: Identification of differentially methylated genes associated with cetuximab and erlotinib resistance

Abstract Anti-EGFR therapy is among the most promising molecular targeted therapies against cancer developed in the past decade. Two classes of EGFR-directed therapeutic inhibitors are currently in clinical use: anti-EGFR monoclonal antibodies, such as cetuximab, and small-molecule tyrosine kinase inhibitors (TKIs) of EGFR, such as erlotinib. Although both classes of drugs are capable of inducing an initial therapeutic response, primary as well as acquired drug resistance is frequently observed. Emerging evidence has linked epigenetic changes, such as DNA methylation at CpG islands, to the development of cytotoxic chemotherapeutic drug resistance. To identify genes that are specifically methylated during the evolution of resistance to anti-EGFR therapeutic agents, we employed two parental NSCLC and HNSCC cell lines and their resistant derivatives to either erlotinib or cetuximab. DNA from each pair was used for methylation-specific array containing a panel of 64 genes that are commonly known to be regulated through promoter methylation. We found that death-associated protein kinase 1 (DAPK1) was hypermethylated in drug resistant cells generated from both parental cell lines. Restoration of DAPK1 into the resistant cells by stable transfection re-sensitized the cells to both erlotinib and cetuximab. Conversely, siRNA-mediated knockdown of DAPK1 induced resistance in the parental sensitive cells. These results demonstrate that DAPK1 plays an important role in both cetuximab and erlotinib resistance in NSCLC and HNSCC cancer cells, and that promoter methylation of its gene frequently occurs during the chronic treatment of anti-EGFR agents. In conclusion, DAPK1 could be a novel target for the improvement of therapeutic benefit of anti-EGFR therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1892. doi:1538-7445.AM2012-1892

Neratinib Reverses ATP-Binding Cassette B1-Mediated Chemotherapeutic Drug Resistance In Vitro, In Vivo, and Ex Vivo

Neratinib, an irreversible inhibitor of epidermal growth factor receptor and human epidermal receptor 2, is in phase III clinical trials for patients with human epidermal receptor 2-positive, locally advanced or metastatic breast cancer. The objective of this study was to explore the ability of neratinib to reverse tumor multidrug resistance attributable to overexpression of ATP-binding cassette (ABC) transporters. Our results showed that neratinib remarkably enhanced the sensitivity of ABCB1-overexpressing cells to ABCB1 substrates. It is noteworthy that neratinib augmented the effect of chemotherapeutic agents in inhibiting the growth of ABCB1-overexpressing primary leukemia blasts and KBv200 cell xenografts in nude mice. Furthermore, neratinib increased doxorubicin accumulation in ABCB1-overexpressing cell lines and Rhodamine 123 accumulation in ABCB1-overexpressing cell lines and primary leukemia blasts. Neratinib stimulated the ATPase activity of ABCB1 at low concentrations but inhibited it at high concentrations. Likewise, neratinib inhibited the photolabeling of ABCB1 with [(125)I]iodoarylazidoprazosin in a concentration-dependent manner (IC(50) = 0.24 μM). Neither the expression of ABCB1 at the mRNA and protein levels nor the phosphorylation of Akt was affected by neratinib at reversal concentrations. Docking simulation results were consistent with the binding conformation of neratinib within the large cavity of the transmembrane region of ABCB1, which provides computational support for the cross-reactivity of tyrosine kinase inhibitors with human ABCB1. In conclusion, neratinib can reverse ABCB1-mediated multidrug resistance in vitro, ex vivo, and in vivo by inhibiting its transport function.

Biological Role of Aldo–Keto Reductases in Retinoic Acid Biosynthesis and Signaling

Several aldo–keto reductase (AKR) enzymes from subfamilies 1B and 1C show retinaldehyde reductase activity, having low Km and kcat values. Only AKR1B10 and 1B12, with all-trans-retinaldehyde, and AKR1C3, with 9-cis-retinaldehyde, display high catalytic efficiency. Major structural determinants for retinaldehyde isomer specificity are located in the external loops (A and C for AKR1B10, and B for AKR1C3), as assessed by site-directed mutagenesis and molecular dynamics. Cellular models have shown that AKR1B and 1C enzymes are well suited to work in vivo as retinaldehyde reductases and to regulate retinoic acid (RA) biosynthesis at hormone pre-receptor level. An additional physiological role for the retinaldehyde reductase activity of these enzymes, consistent with their tissue localization, is their participation in β-carotene absorption. Retinaldehyde metabolism may be subjected to subcellular compartmentalization, based on enzyme localization. While retinaldehyde oxidation to RA takes place in the cytosol, reduction to retinol could take place in the cytosol by AKRs or in the membranes of endoplasmic reticulum by microsomal retinaldehyde reductases. Upregulation of some AKR1 enzymes in different cancer types may be linked to their induction by oxidative stress and to their participation in different signaling pathways related to cell proliferation. AKR1B10 and AKR1C3, through their retinaldehyde reductase activity, trigger a decrease in the RA biosynthesis flow, resulting in RA deprivation and consequently lower differentiation, with an increased cancer risk in target tissues. Rational design of selective AKR inhibitors could lead to development of novel drugs for cancer treatment as well as reduction of chemotherapeutic drug resistance.