Temozolomide-modulated glioma proteome: Role of interleukin-1 receptor-associated kinase-4 (IRAK4) in chemosensitivity

The current treatment for glioblastoma includes temozolomide (TMZ) chemotherapy, yet the mechanism of action of TMZ is not thoroughly understood. Here, we investigated the TMZ-induced changes in the proteome of the glioma-derived cell line (U251) by 2D DIGE. We found 95 protein spots to be significantly altered in their expression after TMZ treatment. MS identified four upregulated spots: aspartyl tRNA synthetase glutathione synthetase, interleukin-1 receptor-associated kinase-4 (IRAK4), and breast carcinoma amplified sequence-1 and one downregulated spot: optineurin. TMZ-induced regulation of these five genes was validated by RT-qPCR andWestern blot analysis. RNAi-mediated knockdown of IRAK4, an important mediator of Toll-like receptors signaling and chemoresistance, rendered the glioma cells resistant to TMZ. High levels of IRAK4 induced upon TMZ treatment resulted in IRAK1 downregulation and inhibition of NFkB pathway. Endogenous IRAK4 protein, but not transcript levels in glioma cell lines, correlated with TMZ sensitivity. Thus, we have identified several TMZ-modulated proteins and discovered an important novel role for IRAK4 in determining TMZ sensitivity of glioma cells through its ability to inhibit Toll-like receptor signaling and NFkB pathway.

HIF-1alpha role in oxygen-dependent radio- and chemosensitivity

Poor oxygenation (hypoxia) is a common characteristic of human solid tumours, and is associated with cell survival, metastasis and resistance to radio- and chemotherapies. Hypoxia-induced stabilisation of hypoxia-inducible factor-1α (HIF-1α) leads to changes in expression of various genes associated with growth, vascularisation and metabolism. However whether HIF-1α plays a causal role in promoting hypoxic resistance to antitumour therapies remains unclear. In this study we used pharmacological and genetic methods to investigate the HIF-1α contribution to radio- and chemoresistance in four cancer cell lines derived from cervical, breast, prostate and melanoma human tumours. Under normoxia or hypoxia (<0.2% or 0.5% oxygen) the cells were exposed to either a standard irradiation dose (6.2 Gy) or chemotherapeutic drug (cisplatin), and subsequent cell proliferation (after 7 days) was measured in terms of resazurin reduction. Oxygen-dependent radio- and chemosensitivity was evident in all wild type whereas it was reduced or abolished in HIF-1α (siRNA) knockdown cells. The effects of HIF-1α-modulating drugs (EDHB, CoCl2, deferoxamine to stabilise and R59949 to destabilise it) reflected both HIF-1α-dependent and independent mechanisms. Collectively the data show that HIF-1α played a causal role in our in vitro model of hypoxia-induced radioresistance whereas its contribution to oxygendependent sensitivity to cisplatin was less clear-cut. Although this behavior is likely to be conditioned by further biological and physical factors operating in vivo, it is consistent with the hypothesis that interventions directed at HIF-1α may improve the clinical effectiveness of tumour treatments.

Manganese Superoxide Dismutase Is a Promising Target for Enhancing Chemosensitivity of Basal-Like Breast Carcinoma

AIMS: Although earlier reports highlighted a tumor suppressor role for manganese superoxide dismutase (MnSOD), recent evidence indicates increased expression in a variety of human cancers including aggressive breast carcinoma. In the present article, we hypothesized that MnSOD expression is significantly amplified in the aggressive breast carcinoma basal subtype, and targeting MnSOD could be an attractive strategy for enhancing chemosensitivity of this highly aggressive breast cancer subtype.

RESULTS: Using MDA-MB-231 and BT549 as a model of basal breast cancer cell lines, we show that knockdown of MnSOD decreased the colony-forming ability and sensitized the cells to drug-induced cell death, while drug resistance was associated with increased MnSOD expression. In an attempt to develop a clinically relevant approach to down-regulate MnSOD expression in patients with basal breast carcinoma, we employed activation of the peroxisome proliferator-activated receptor gamma (PPARγ) to repress MnSOD expression; PPARγ activation significantly reduced MnSOD expression, increased chemosensitivity, and inhibited tumor growth. Moreover, as a proof of concept for the clinical use of PPARγ agonists to decrease MnSOD expression, biopsies derived from breast cancer patients who had received synthetic PPARγ ligands as anti-diabetic therapy had significantly reduced MnSOD expression. Finally, we provide evidence to implicate peroxynitrite as the mechanism involved in the increased sensitivity to chemotherapy induced by MnSOD repression.

INNOVATION AND CONCLUSION: These data provide evidence to link increased MnSOD expression with the aggressive basal breast cancer, and underscore the judicious use of PPARγ ligands for specifically down-regulating MnSOD to increase the chemosensitivity of this subtype of breast carcinoma.

Interleukin-8 is associated with proliferation, migration, angiogenesis and chemosensitivity in vitro and in vivo in colon cancer cell line models

Interleukin-8 (IL-8), a chemokine with a defining CXC amino acid motif, is known to possess tumorigenic and proangiogenic properties. Overexpression of IL-8 has been detected in many human tumors, including colorectal cancer (CRC), and is associated with poor prognosis. The goal of our study was to determine the role of IL-8 overexpression in CRC cells in vitro and in vivo. We stably transfected the IL-8 cDNA into two human colon cancer cell lines, HCT116 and Caco2, and selected IL-8-secreting transfectants. Real-time RT-PCR confirmed that IL-8 mRNA was overexpressed in IL-8 transfectants with 45- to 85-fold higher than parental cells. The IL-8-transfected clones secreted 19- to 28-fold more IL-8 protein than control and parental cells as detected by ELISA. The IL-8 transfectants demonstrated increased cellular proliferation, cell migration and invasion based on functional assays. Growth inhibition studies showed that IL-8 overexpression lead to a significant resistance to oxaliplatin (p < 0.0001). Inhibition of IL-8 overexpression with small interfering RNA reversed the observed increases in tumorigenic functions and oxaliplatin resistance, suggesting that IL-8 not only provides a proliferative advantage but also promotes the metastatic potential of colon cancer cells. Using a tumor xenograft model, IL-8-expressing cells formed significantly larger tumors than the control cells with increased microvessel density. Together, these findings indicate that overexpression of IL-8 promotes tumor growth, metastasis, chemoresistance and angiogenesis, implying IL-8 to be an important therapeutic target in CRC.

The locus coeruleus and central chemosensitivity

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Inhibition of nuclear factor-kappa B by dehydroxymethylepoxyquinomicin induces schedule-dependent chemosensitivity to anticancer drugs and enhances chemoinduced apoptosis in osteosarcoma cells

Osteosarcoma (OS) is the most common primary malignant bone tumor, usually developing in children and adolescents, and is highly invasive and metastatic, potentially developing chemoresistance. Thus, novel effective treatment regimens are urgently needed. This study was the first to investigate the anticancer effects of dehydroxymethylepoxyquinomicin (DHMEQ), a highly specific nuclear factor-kappa B (NF-kappa B) inhibitor, on the OS cell lines HOS and MG-63. We demonstrate that NF-kappa B blockade by DHMEQ inhibits proliferation, decreases the mitotic index, and triggers apoptosis of OS cells. We examined the effects of combination treatment with DHMEQ and cisplatin, doxorubicin, or methotrexate, drugs commonly used in OS treatment. Using the median effect method of Chou and Talalay, we evaluated the combination indices for simultaneous and sequential treatment schedules. In all cases, combination with a chemotherapeutic drug produced a synergistic effect, even at low single-agent cytotoxic levels. When cells were treated with DHMEQ and cisplatin, a more synergistic effect was obtained using simultaneous treatment. For the doxorubicin and methotrexate combination, a more synergistic effect was achieved with sequential treatment using DHMEQ before chemotherapy. These synergistic effects were accompanied by enhancement of chemoinduced apoptosis. Interestingly, the highest apoptotic effect was reached with sequential exposure in both cell lines, independent of the chemotherapeutic agent used. Likewise, DHMEQ decreased cell invasion and migration, crucial steps for tumor progression. Our data suggest that combining DHMEQ with chemotherapeutic drugs might be useful for planning new therapeutic strategies for OS treatment, mainly in resistant and metastatic cases. Anti-Cancer Drugs 23:638-650 (C) 2012 Wolters Kluwer Health broken vertical bar Lippincott Williams & Wilkins.

Inhibition of Aurora kinases enhances chemosensitivity to temozolomide and causes radiosensitization in glioblastoma cells

Glioblastoma remains one of the most devastating human malignancies, and despite therapeutic advances, there are no drugs that significantly improve the patient survival. Altered expression of the Aurora kinases was found in different malignancies, and their inhibition has been studied in cancer therapy. In this study, we analyzed the expression of Aurora A and Aurora B in glioblastoma samples and also analyzed whether the effects of Aurora kinase inhibition were associated with temozolomide or not on cell lines and primary cultures of glioblastoma. RT-PCR assays were used to determine the mRNA expression in glioblastoma tumor samples and in the cell lines. Cell proliferation was measured by XTT assay, and apoptosis was determined by flow cytometry. Drug combination analyses were made based in Chou-Talalay method. Gamma radiation for clonogenic survival used the doses of 2, 4 and 6 Gy. Changes in Aurora B level were assessed by Western blot analysis. Aurora A and B were expressed in glioblastoma samples as well as in the glioblastoma cell lines (n = 6). Moreover, ZM447439, a selective Aurora kinase inhibitor, decreased the proliferation separately and synergistically with temozolomide in primary cultures and cell lines of glioblastoma. ZM also enhanced the effects of radiation on the two cell lines studied (U343 and U251), mainly when associated with TMZ in U343 cells. Treatment with ZM induced apoptotic cell death and diminished Aurora B protein level. These data suggest that Aurora kinase inhibition may be a target for glioblastoma treatment and could be used as adjuvant to chemo- and radiotherapy.

Induction of apoptosis and enhancement of chemosensitivity in human prostate cancer LNCaP cells using bispecific antisense oligonucleotide targeting Bcl-2 and Bcl-xL genes

OBJECTIVE: To determine whether a specifically designed bispecific (Bcl-2/Bcl-xL) antisense oligonucleotide (ASO) induces apoptosis and enhances chemosensitivity in human prostate cancer LNCaP cells, as Bcl-2 and Bcl-xL are both anti-apoptotic genes associated with treatment resistance and tumour progression in many malignancies, including prostate cancer. MATERIALS AND METHODS: Inhibition of Bcl-2 and Bcl-xL expression by the bispecific ASO was evaluated using real-time reverse transcription-polymerase chain reaction and Western blotting, while growth inhibition and induction of apoptosis were analysed by a crystal violet assay, flow cytometry and Western blotting of apoptosis-relevant proteins. The effect of combined treatment with bispecific ASO and chemotherapy or small-interference RNA (siRNA) targeting the clusterin gene was also investigated. RESULTS: Bispecific ASO reduced Bcl-2 and Bcl-xL expression in LNCaP cells in a dose-dependent manner. There was cell growth inhibition, increases in the sub-G0-G1 fraction, and cleavage of caspase-3 and poly(ADP-Ribose) polymerase proteins in LNCaP cells after bispecific ASO treatment. Interestingly, Bcl-2/Bcl-xL bispecific ASO treatment also resulted in the down-regulation of Mcl-1 and up-regulation of Bax. The sensitivity of LNCaP cells to mitoxantrone, docetaxel or paclitaxel was significantly increased, reducing the 50% inhibitory concentration by 45%, 80% or 90%, respectively. Furthermore, the apoptotic induction by Bcl-2/Bcl-xL bispecific ASO was synergistically enhanced by siRNA-mediated inhibition of clusterin, a cytoprotective chaperone that interacts with and inhibits activated Bax. CONCLUSIONS: These findings support the concept of the targeted suppression of Bcl-2 anti-apoptotic family members using multitarget inhibition strategies for prostate cancer, through the effective induction of apoptosis.

Empirical chemosensitivity testing in a spheroid model of ovarian cancer using a microfluidics-based multiplex platform.

The use of biomarkers to infer drug response in patients is being actively pursued, yet significant challenges with this approach, including the complicated interconnection of pathways, have limited its application. Direct empirical testing of tumor sensitivity would arguably provide a more reliable predictive value, although it has garnered little attention largely due to the technical difficulties associated with this approach. We hypothesize that the application of recently developed microtechnologies, coupled to more complex 3-dimensional cell cultures, could provide a model to address some of these issues. As a proof of concept, we developed a microfluidic device where spheroids of the serous epithelial ovarian cancer cell line TOV112D are entrapped and assayed for their chemoresponse to carboplatin and paclitaxel, two therapeutic agents routinely used for the treatment of ovarian cancer. In order to index the chemoresponse, we analyzed the spatiotemporal evolution of the mortality fraction, as judged by vital dyes and confocal microscopy, within spheroids subjected to different drug concentrations and treatment durations inside the microfluidic device. To reflect microenvironment effects, we tested the effect of exogenous extracellular matrix and serum supplementation during spheroid formation on their chemotherapeutic response. Spheroids displayed augmented chemoresistance in comparison to monolayer culturing. This resistance was further increased by the simultaneous presence of both extracellular matrix and high serum concentration during spheroid formation. Following exposure to chemotherapeutics, cell death profiles were not uniform throughout the spheroid. The highest cell death fraction was found at the center of the spheroid and the lowest at the periphery. Collectively, the results demonstrate the validity of the approach, and provide the basis for further investigation of chemotherapeutic responses in ovarian cancer using microfluidics technology. In the future, such microdevices could provide the framework to assay drug sensitivity in a timeframe suitable for clinical decision making.

Towards personalized medicine: Chemosensitivity assays of patient lung cancer cell spheroids in a perfused microfluidic platform

Cancer is responsible for millions of deaths worldwide and the variability in disease patterns calls for patient-specific treatment. Therefore, personalized treatment is expected to become a daily routine in prospective clinical tests. In addition to genetic mutation analysis, predictive chemosensitive assays using patient's cells will be carried out as a decision making tool. However, prior to their widespread application in clinics, several challenges linked to the establishment of such assays need to be addressed. To best predict the drug response in a patient, the cellular environment needs to resemble that of the tumor. Furthermore, the formation of homogeneous replicates from a scarce amount of patient's cells is essential to compare the responses under various conditions (compound and concentration). Here, we present a microfluidic device for homogeneous spheroid formation in eight replicates in a perfused microenvironment. Spheroid replicates from either a cell line or primary cells from adenocarcinoma patients were successfully created. To further mimic the tumor microenvironment, spheroid co-culture of primary lung cancer epithelial cells and primary pericytes were tested. A higher chemoresistance in primary co-culture spheroids compared to primary monoculture spheroids was found when both were constantly perfused with cisplatin. This result is thought to be due to the barrier created by the pericytes around the tumor spheroids. Thus, this device can be used for additional chemosensitivity assays (e.g. sequential treatment) of patient material to further approach the personalized oncology field.

Selective induction of p53 and chemosensitivity in RB-deficient cells by E1A mutants unable to bind the RB-related proteins

The adenovirus E1A oncoprotein renders primary cells sensitive to the induction of apoptosis by diverse stimuli, including many anticancer agents. E1A-expressing cells accumulate p53 protein, and p53 potentiates drug-induced apoptosis. To determine how E1A promotes chemosensitivity, a series of E1A mutants were introduced into primary human and mouse fibroblasts using high-titer recombinant retroviruses, allowing analysis of E1A in genetically normal cells outside the context of adenovirus infection. Mutations that disrupted apoptosis and chemosensitivity separated into two complementation groups, which correlated precisely with the ability of E1A to associate with either the p300/CBP or retinoblastoma protein families. Furthermore, E1A mutants incapable of binding RB, p107, and p130 conferred chemosensitivity to fibroblasts derived from RB-deficient mice, but not fibroblasts from mice lacking p107 or p130. Hence, inactivation of RB, but not p107 or p130, is required for chemosensitivity induced by E1A. Finally, the same E1A functions that promote drug-induced apoptosis also induce p53. Together, these data demonstrate that p53 accumulation and chemosensitivity are linked to E1A’s oncogenic potential, and identify a strategy to selectively induce apoptosis in RB-deficient tumor cells.