Chemotherapeutic resistance in anaplastic astrocytoma cell lines treated with a temozolomide–lomeguatrib combination

The treatment of anaplastic astrocytoma (AA) is controversial. New chemotherapeutic approaches are needed for AA treatment. Temozolomide (TMZ) is one of the chemotherapeutic drugs for the treatment of AA. The cytotoxic effects of TMZ can be removed by the MGMT (O(6)-methylguanine-DNA methyltransferase) enzyme. Then, chemotherapeutic resistance to TMZ occurs. MGMT inhibition by MGMT inactivators (such as lomeguatrib) is an important anticancer therapeutic approach to circumvent TMZ resistance. We aim to investigate the effect of TMZ–lomeguatrib combination on MGMT expression and TMZ sensitivity of SW1783 and GOS-3 AA cell lines. The sensitivity of SW1783 and GOS-3 cell lines to TMZ and to the combination of TMZ and lomeguatrib was determined by a cytotoxicity assay. MGMT methylation was detected by MS-PCR. MGMT and p53 expression were investigated by real-time PCR after drug treatment, and the proportion of apoptotic cells was analyzed by flow cytometry. When the combination of TMZ–lomeguatrib (50 μM) was used in AA cell lines, IC₅₀ values were reduced compared to only using TMZ. MGMT expression was decreased, p53 expression was increased, and the proportion of apoptotic cells was induced in both cell lines. The lomeguatrib–TMZ combination did not have any effect on the cell cycle and caused apoptosis by increasing p53 expression and decreasing MGMT expression. Our study is a pilot study investigating a new therapeutic approach for AA treatment, but further research is needed.     

Inhibition of Casein Kinase II by CX-4945, But Not Yes-associated protein (YAP) by Verteporfin,Enhances the Antitumor Efficacy of Temozolomide in Glioblastoma

Overcoming temozolomide (TMZ) resistance in glioma cancer cells remains a major challenge to the effective treatment of the disease. Increasing TMZ efficacy for patients with glioblastoma (GBM) is urgently needed because TMZ treatment is the standard chemotherapy protocol for adult patients with glioblastoma. O⁶-methylguanine-DNA-methyltransferase (MGMT) overexpression is associated with TMZ resistance, and low MGMT is a positive response marker for TMZ therapy. Here, we used 3 glioma cell lines (SF767, U373, and LN229), which had different levels of TMZ sensitivity. We found TMZ sensitivity is positively correlated with MGMT expression and multidrug-resistance protein ABC subfamily G member 2 (ABCG2) in these cells. CK2-STAT3 signaling and Hippo-YAP signaling are reported to regulate MGMT expression and ABCG2 expression, respectively. We combined CK2 inhibitor CX-4945 and YAP inhibitor verteporfin with TMZ treatment. We found that CX-4945 but not verteporfin can sensitize TMZ-resistant cells SF767 to TMZ and that CX-4945 and TMZ combinational treatment was effective for glioma treatment in mouse models compared with TMZ alone.ImplicationsA combination of CK2 inhibitor with TMZ may improve the therapeutic efficiency of TMZ toward GBM with acquired resistance.     

Establishment and Biological Characterization of a Panel of Glioblastoma Multiforme (GBM) and GBM Variant Oncosphere Cell Lines

Objective

Human tumor cell lines form the basis of the majority of present day laboratory cancer research. These models are vital to studying the molecular biology of tumors and preclinical testing of new therapies. When compared to traditional adherent cell lines, suspension cell lines recapitulate the genetic profiles and histologic features of glioblastoma multiforme (GBM) with higher fidelity. Using a modified neural stem cell culture technique, here we report the characterization of GBM cell lines including GBM variants.

Methods

Tumor tissue samples were obtained intra-operatively and cultured in neural stem cell conditions containing growth factors. Tumor lines were characterized in vitro using differentiation assays followed by immunostaining for lineage-specific markers. In vivo tumor formation was assayed by orthotopic injection in nude mice. Genetic uniqueness was confirmed via short tandem repeat (STR) DNA profiling.

Results

Thirteen oncosphere lines derived from GBM and GBM variants, including a GBM with PNET features and a GBM with oligodendroglioma component, were established. All unique lines showed distinct genetic profiles by STR profiling. The lines assayed demonstrated a range of in vitro growth rates. Multipotency was confirmed using in vitro differentiation. Tumor formation demonstrated histologic features consistent with high grade gliomas, including invasion, necrosis, abnormal vascularization, and high mitotic rate. Xenografts derived from the GBM variants maintained histopathological features of the primary tumors.

Conclusions

We have generated and characterized GBM suspension lines derived from patients with GBMs and GBM variants. These oncosphere cell lines will expand the resources available for preclinical study.     

NETRIN-4 Protects Glioblastoma Cells FROM Temozolomide Induced Senescence

Glioblastoma multiforme is the most common primary tumor of the central nervous system. The drug temozolomide (TMZ) prolongs lifespan in many glioblastoma patients. The sensitivity of glioblastoma cells to TMZ is interfered by many factors, such as the expression of O-6-methylguanine-DNA methyltransferase (MGMT) and activation of AKT signaling. We have recently identified the interaction between netrin-4 (NTN4) and integrin beta-4 (ITGB4), which promotes glioblastoma cell proliferation via activating AKT-mTOR signaling pathway. In the current work we have explored the effect of NTN4/ITGB4 interaction on TMZ induced glioblastoma cell senescence. We report here that the suppression of either ITGB4 or NTN4 in glioblastoma cell lines significantly enhances cellular senescence. The sensitivity of GBM cells to TMZ was primarily determined by the expression of MGMT. To omit the effect of MGMT, we concentrated on the cell lines devoid of expression of MGMT. NTN4 partially inhibited TMZ induced cell senescence and rescued AKT from dephosphorylation in U251MG cells, a cell line bearing decent levels of ITGB4. However, addition of exogenous NTN4 displayed no significant effect on TMZ induced senescence rescue or AKT activation in U87MG cells, which expressed ITGB4 at low levels. Furthermore, overexpression of ITGB4 combined with exogenous NTN4 significantly attenuated U87MG cell senescence induced by TMZ. These data suggest that NTN4 protects glioblastoma cells from TMZ induced senescence, probably via rescuing TMZ triggered ITGB4 dependent AKT dephosphorylation. This suggests that interfering the interaction between NTN4 and ITGB4 or concomitant use of the inhibitors of the AKT pathway may improve the therapeutic efficiency of TMZ.     

An Individual Patient Data Meta-Analysis on Characteristics,Treatments and Outcomes of Glioblastoma/ Gliosarcoma Patients with Metastases Outside of the Central Nervous System

Purpose

To determine the characteristics, treatments and outcomes of patients with glioblastoma multiforme (GBM) or gliosarcoma (GS) and metastases outside of the central nervous system (CNS).

Methods

PubMed and Web of Science searches for peer-reviewed articles pertaining to GBM/ GS patients with metastatic dissemination were conducted using the keywords gliosarcoma, glioblastoma, GBM, metastasis, metastases and metastatic. Additionally, we performed hand search following the references from the selected papers. Cases with metastases to the CNS were excluded and evaluated in a separate study.

Results

109 articles published between 1928 and 2013 were eligible. They reported on 150 patients. We observed a remarkable increase in the number of cases per decade over time. Median overall survival from diagnosis of metastasis (OS_M+) was 6.0 ± 0.8 months and median overall survival from initial diagnosis (OS_ID) 13 ± 2.4 months. On univariate analyses, gender, age, the histological subtype, the time interval between initial diagnosis and diagnosis of metastasis and pulmonary involvement did not influence OS_M+. We did not observe any substantial treatment progress. A comparison of the present cohort with 84 GBM/ GS patients with exclusive CNS dissemination suggests that metastases outside the CNS are related to a slightly more favorable outcome.

Conclusions

The occurrence of extra-CNS metastasis from GBM/ GS is associated with a dismal prognosis, however it seems to compare slightly favorable to CNS dissemination. Crucial treatment progress has not been achieved over recent decades. A central registry should be considered to consecutively gain more information about the ideal therapeutic approach.     

Resistance to Bleomycin in Cancer Cell Lines Is Characterized by Prolonged Doubling Time,Reduced DNA Damage and Evasion of G2/M Arrest and Apoptosis

Background

To establish, characterize and elucidate potential mechanisms of acquired bleomycin (BLM) resistance using human cancer cell lines. Seven BLM-resistant cell lines were established by exposure to escalating BLM concentrations over a period of 16-24 months. IC₅₀ values and cell doubling times were quantified using a real time cytotoxicity assay. COMET and γ-H2AX assays, cell cycle analysis, and apoptosis assessment further investigated the mechanisms of BLM resistance in these cell lines.

Results

Compared with parental cell lines, real time cytotoxicity assays revealed 7 to 49 fold increases in IC₅₀ and a mean doubling time increase of 147 % (range 64 %-352%) in BLM-resistant sub-clones (p<0.05 for both). Higher maintenance BLM concentrations were associated with higher IC₅₀ and increased doubling times (p<0.05). Significantly reduced DNA damage (COMET and γ-H2AX assays), G2/M arrest, and apoptosis (p<0.05 for each set of comparison) following high-dose acute BLM exposure was observed in resistant sub-clones, compared with their BLM-sensitive parental counterparts. Three weeks of BLM-free culturing resulted in a partial return to BLM sensitivity in 3/7 BLM-resistant sub-clones (p<0.05).

Conclusion

Bleomycin resistance may be associated with reduced DNA damage after bleomycin exposure, resulting in reduced G2/M arrest, and reduced apoptosis.     

The Predictive but Not Prognostic Value of MGMT Promoter Methylation Status in Elderly Glioblastoma Patients: A Meta-Analysis

Background

The clinical implication of O6-methylguanine-DNA methyltransferase (MGMT) promoter status is ill-defined in elderly glioblastoma patients. Here we report a meta-analysis to seek valid evidence for its clinical relevance in this subpopulation.

Methods

Literature were searched and reviewed in a systematic manner using the PubMed, EMBASE and Cochrane databases. Studies investigating the association between MGMT promoter status and survival data of elderly patients (≥65 years) were eligible for inclusion.

Results

Totally 16 studies were identified, with 13 studies included in the final analyses. The aggregate proportion of MGMT promoter methylation in elderly patients was 47% (95% confidence interval [CI]: 42–52%), which was similar to the value for younger patients. The analyses showed differential effects of MGMT status on overall survival (OS) of elderly patients according to assigned treatments: methylated vs. unmethylated: (1) temozolomide (TMZ)-containing therapies: hazard ratio [HR] 0.49, 95% CI 0.41–0.58; (2) TMZ-free therapies: HR 0.97, 95% CI 0.77–1.21. More importantly, a useful predictive value was observed by an interaction analysis: TMZ-containing therapies vs. RT alone: (1) methylated tumors: HR 0.48, 95% CI 0.36–0.65; (2) unmethylated tumors: HR 1.14; 95% CI 0.90–1.44.

Conclusion

The meta-analysis reports an age-independent presence of MGMT promoter methylation. More importantly, the study encouraged routine testing of MGMT promoter status especially in elderly glioblastoma patients by indicating a direct linkage between biomarker test and individual treatment decision. Future studies are needed to justify the mandatory testing in younger patients.     

Effectiveness of interferon-beta and temozolomide combination therapy against temozolomide-refractory recurrent anaplastic astrocytoma

Background

Malignant gliomas recur even after extensive surgery and chemo-radiotherapy. Although a relatively novel chemotherapeutic agent, temozolomide (TMZ), has demonstrated promising activity against recurrent glioma, the effects last only a few months and drug resistance develops thereafter in most cases. Induction of O⁶-methylguanine-DNA methyltransferase (MGMT) in tumors is considered to be responsible for resistance to TMZ. Interferon-beta has been reported to suppress MGMT in an experimental glioma model. Here we report a patient with TMZ-refractory anaplastic astrocytoma (AA) who was treated successfully with a combination of interferon-beta and TMZ.

Case presentation

A patient with recurrent AA after radiation-chemotherapy and stereotactic radiotherapy was treated with TMZ. After 6 cycles, the tumor became refractory to TMZ, and the patient was treated with interferon-beta at 3 × 10⁶ international units/body, followed by 5 consecutive days of 200 mg/m² TMZ in cycles of 28 days. After the second cycle the tumor decreased in size by 50% (PR). The tumor showed further shrinkage after 8 months and the patient's KPS improved from 70% to 100%. The immunohistochemical study of the initial tumor specimen confirmed positive MGMT protein expression.

Conclusion

It is considered that interferon-beta pre-administration increased the TMZ sensitivity of the glioma, which had been refractory to TMZ monotherapy.

     

Inhibition of Elongation Factor-2 Kinase Augments the Antitumor Activity of Temozolomide against Glioma

Background

Glioblastoma multiforme (GBM), the most common form of brain cancer with an average survival of less than 12 months, is a highly aggressive and fatal disease characterized by survival of glioma cells following initial treatment, invasion through the brain parenchyma and destruction of normal brain tissues, and ultimately resistance to current treatments. Temozolomide (TMZ) is commonly used chemotherapy for treatment of primary and recurrent high-grade gliomas. Nevertheless, the therapeutic outcome of TMZ is often unsatisfactory. In this study, we sought to determine whether eEF-2 kinase affected the sensitivity of glioma cells to treatment with TMZ.

Methodology/Principal Findings

Using RNA interference approach, a small molecule inhibitor of eEF-2 kinase, and in vitro and in vivo glioma models, we observed that inhibition of eEF-2 kinase could enhance sensitivity of glioma cells to TMZ, and that this sensitizing effect was associated with blockade of autophagy and augmentation of apoptosis caused by TMZ.

Conclusions/Significance

These findings demonstrated that targeting eEF-2 kinase can enhance the anti-glioma activity of TMZ, and inhibitors of this kinase may be exploited as chemo-sensitizers for TMZ in treatment of malignant glioma.     

Cinnamomum cassia Suppresses Caspase-9 through Stimulation of AKT1 in MCF-7 Cells but Not in MDA-MB-231 Cells

Background

Cinnamomum cassia bark is a popular culinary spice used for flavoring and in traditional medicine. C. cassia extract (CE) induces apoptosis in many cell lines. In the present study, particular differences in the mechanism of the anti-proliferative property of C. cassia on two breast cancer cell lines, MCF-7 and MDA-MB-231, were elucidated.

Methodology/Principal Findings

The hexane extract of C. cassia demonstrated high anti-proliferative activity against MCF-7 and MDA-MB-231 cells (IC₅₀, 34±3.52 and 32.42 ±0.37 μg/ml, respectively). Oxidative stress due to disruption of antioxidant enzyme (SOD, GPx and CAT) activity is suggested as the probable cause for apoptosis initiation. Though the main apoptosis pathway in both cell lines was found to be through caspase-8 activation, caspase-9 was also activated in MDA-MB-231 cells but suppressed in MCF-7 cells. Gene expression studies revealed that AKT1, the caspase-9 suppressor, was up-regulated in MCF-7 cells while down-regulated in MDA-MB-231 cells. Although, AKT1 protein expression in both cell lines was down-regulated, a steady increase in MCF-7 cells was observed after a sharp decrease of suppression of AKT1. Trans-cinnamaldehyde and coumarin were isolated and identified and found to be mainly responsible for the observed anti-proliferative activity of CE (Cinnamomum cassia).

Conclusion

Activation of caspase-8 is reported for the first time to be involved as the main apoptosis pathway in breast cancer cell lines upon treatment with C. cassia. The double effects of C. cassia on AKT1 gene expression in MCF-7 cells is reported for the first time in this study.     

Chemotherapeutic effect of a novel temozolomide analog on nasopharyngeal carcinoma in vitro and in vivo

Background

Many patients with nasopharyngeal carcinoma (NPC) face poor prognosis. Due to its hidden anatomical location, the tumor is usually diagnosed quite late, and despite initially successful treatment with radiation and cisplatin, many patients will relapse and succumb to the disease. New treatment options are urgently needed. We have performed preclinical studies to evaluate the potential NPC therapeutic activity of a newly developed analog of temozolomide (TMZ), an alkylating agent that is the current chemotherapeutic standard of care for patients with malignant glioma.

Results

TMZ was covalently conjugated to the natural monoterpene perillyl alcohol (POH), creating the novel fusion compound NEO212. Its impact on two NPC cell lines was studied through colony formation assays, cell death ELISA, immunoblots, and in vivo testing in tumor-bearing mice. In vitro, NEO212 effectively triggered tumor cell death, and its potency was significantly greater than that of its individual components, TMZ or POH alone. Intriguingly, merely mixing TMZ with POH also was unable to achieve the superior potency of the conjugated compound NEO212. Treatment of NPC cells with NEO212 inactivated the chemoprotective DNA repair protein MGMT (O6-methylguanine methyltransferase), resulting in significant chemosensitization of cells to a second round of drug treatment. When tested in vivo, NEO212 reduced tumor growth in treated animals.

Conclusion

Our results demonstrate anticancer activity of NEO212 in preclinical NPC models, suggesting that this novel compound should be evaluated further for the treatment of patients with NPC.

Electronic supplementary material

The online version of this article (doi:10.1186/s12929-015-0175-6) contains supplementary material, which is available to authorized users.     

The Reversal Effects of 3-Bromopyruvate on Multidrug Resistance In Vitro and In Vivo Derived from Human Breast MCF-7/ADR Cells

Purpose

P-glycoprotein mediated efflux is one of the main mechanisms for multidrug resistance in cancers, and 3-Bromopyruvate acts as a promising multidrug resistance reversal compound in our study. To test the ability of 3-Bromopyruvate to overcome P-glycoprotein-mediated multidrug resistance and to explore its mechanisms of multidrug resistance reversal in MCF-7/ADR cells, we evaluate the in vitro and in vivo modulatory activity of this compound.

Methods

The in vitro and in vivo activity was determined using the MTT assay and human breast cancer xenograft models. The gene and protein expression of P-glycoprotein were determined using real-time polymerase chain reaction and the Western blotting technique, respectively. ABCB-1 bioactivity was tested by fluorescence microscopy, multi-mode microplate reader, and flow cytometry. The intracellular levels of ATP, HK-II, and ATPase activity were based on an assay kit according to the manufacturer’s instructions.

Results

3-Bromopyruvate treatment led to marked decreases in the IC₅₀ values of selected chemotherapeutic drugs [e.g., doxorubicin (283 folds), paclitaxel (85 folds), daunorubicin (201 folds), and epirubicin (171 folds)] in MCF-7/ADR cells. 3-Bromopyruvate was found also to potentiate significantly the antitumor activity of epirubicin against MCF-7/ADR xenografts. The intracellular level of ATP decreased 44%, 46% in the presence of 12.5.25 µM 3-Bromopyruvate, whereas the accumulation of rhodamine 123 and epirubicin (two typical P-glycoprotein substrates) in cells was significantly increased. Furthermore, we found that the mRNA and the total protein level of P-glycoprotein were slightly altered by 3-Bromopyruvate. Moreover, the ATPase activity was significantly inhibited when 3-Bromopyruvate was applied.

Conclusion

We demonstrated that 3-Bromopyruvate can reverse P-glycoprotein-mediated efflux in MCF-7/ADR cells. Multidrug resistance reversal by 3-Bromopyruvate occurred through at least three approaches, namely, a decrease in the intracellular level of ATP and HK-II bioactivity, the inhibition of ATPase activity, and the slight decrease in P-glycoprotein expression in MCF-7/ADR cells.     

Brain Radiotherapy plus Concurrent Temozolomide versus Radiotherapy Alone for Patients with Brain Metastases: A Meta-Analysis

Objective

We performed a meta-analysis of randomized clinical trials to compare the efficacy of brain radiotherapy (RT) combined with temozolomide (TMZ) versus RT alone as first-line treatment for brain metastases (BM).

Methods

Medline, Embase, and Pubmed were used to search for relevant randomized controlled trials (RCTs). Two investigators reviewed the abstracts and independently rated the quality of trials and relevant data. The primary outcome was overall survival (OS). Secondary outcomes included progression-free survival (PFS), objective response rate (ORR), and adverse events.

Results

Seven studies were selected from the literature search. RT plus TMZ produced significant improvement in ORR with odds ratio (OR) of 2.27 (95% CI, 1.29 to 4.00; P = 0.005) compared with RT alone. OS and PFS were not significantly different between the two arms (OS: HR, 1.00; P = 0.959; PFS: HR, 0.73; P = 0.232). However, the RT plus TMZ arm was associated with significantly more grade 3 to 4 nausea and thrombocytopenia.

Conclusion

Concomitant RT and TMZ, compared to RT alone, significantly increases ORR in patients with BM, but yields increased toxicity and fails to demonstrate a survival advantage.     

Slug-Dependent Upregulation of L1CAM Is Responsible for the Increased Invasion Potential of Pancreatic Cancer Cells following Long-Term 5-FU Treatment

Background

Pancreatic adenocarcinoma is a lethal disease with 5-year survival of less than 5%. 5-fluorouracil (5-FU) is a principal first-line therapy, but treatment only extends survival modestly and is seldom curative. Drug resistance and disease recurrence is typical and there is a pressing need to overcome this. To investigate acquired 5-FU resistance in pancreatic adenocarcinoma, we established chemoresistant monoclonal cell lines from the Panc 03.27 cell line by long-term exposure to increasing doses of 5-FU.

Results

5-FU-resistant cell lines exhibited increased expression of markers associated with multidrug resistance explaining their reduced sensitivity to 5-FU. In addition, 5-FU-resistant cell lines showed alterations typical for an epithelial-to-mesenchymal transition (EMT), including upregulation of mesenchymal markers and increased invasiveness. Microarray analysis revealed the L1CAM pathway as one of the most upregulated pathways in the chemoresistant clones, and a significant upregulation of L1CAM was seen on the RNA and protein level. In pancreatic cancer, expression of L1CAM is associated with a chemoresistant and migratory phenotype. Using esiRNA targeting L1CAM, or by blocking the extracellular part of L1CAM with antibodies, we show that the increased invasiveness observed in the chemoresistant cells functionally depends on L1CAM. Using esiRNA targeting β-catenin and/or Slug, we demonstrate that in the chemoresistant cell lines, L1CAM expression depends on Slug rather than β-catenin.

Conclusion

Our findings establish Slug-induced L1CAM expression as a mediator of a chemoresistant and migratory phenotype in pancreatic adenocarcinoma cells.