IC50 Values Of Cisplatin Research Articles

Oridonin effectively reverses cisplatin drug resistance in human ovarian cancer cells via induction of cell apoptosis and inhibition of matrix metalloproteinase expression.

Cisplatin is a first generation platinum‑based chemotherapeutic agent, however, the extensive application of cisplatin inevitably results in drug resistance, which is a major obstacle in cancer chemotherapy. The aim of the present study was to investigate the efficiency of reversing cisplatin‑resistance with the use of combination therapy with oridonin and cisplatin in human ovarian cancer cells, and attempt to reduce the side effects of the therapeutic agents when used alone. The half maximal inhibitory concentration (IC50) values of cisplatin were determined in cisplatin‑sensitive and cisplatin‑resistant ovarian cancer cells using an MTT assay. IC50 values of cisplatin in A2780, A2780/DDP, SKOV3 and SKOV3/DDP cells were significantly decreased in a time‑dependent manner. The antitumor effect of oridonin in A2780/DDP cells was also detected by the MTT assay and the inhibitory effects of oridonin increased in a dose‑ and time‑dependent manner. A2780/DDP cells were treated with 20 µM oridonin in combination with increasing concentrations of cisplatin for 48 h, and the result demonstrated that oridonin synergistically increased the antitumor effects of cisplatin in A2780/DDP cells. Notably, the combination treatment of oridonin and cisplatin effectively reversed cisplatin resistance and the IC50 values were significantly decreased from 50.97 µM and 135.20 to 26.12 µM and 73.00 µM in A2780/DDP and SKOV3/DDP cells at 48 h, respectively. Furthermore, oridonin induced cell apoptosis in a dose‑dependent manner and promoted cell‑cycle arrest at the G0/G1 phase in ovarian cancer cells. Oridonin and cisplatin synergistically increased the cell apoptosis rate of A2780/DDP cells, which was detected by fluorescence‑activated cell sorting analysis. Downregulated expression levels of Bcl‑2 and upregulated the expression of Bax protein were demonstrated by western blot analysis, further indicating increased apoptosis. In addition, the expression levels of matrix metalloproteinase (MMP)‑2 and MMP‑9 decreased in a dose‑dependent manner with oridonin treatment. The results from the present study demonstrated that oridonin exerted a synergistic effect with cisplatin to inhibit proliferation and induce cell apoptosis in cisplatin‑resistant ovarian cancer cells. Thus, combination therapy with oridonin and cisplatin effectively reversed cisplatin resistance in human ovarian cancer cells, which may have useful clinical applications.

Abstract A38: Identification through functional genomics screening of factors whose downregulation enhances the side population in ovarian cancer.

Abstract Cancer stem cells (CSC) play a role in chemoresistance and cancer relapse. CSCs are enriched for a side population (SP) of cells as detected by efflux of hoechst dye. In this research, we performed a functional genomics screen to identify genes whose downregulation generates a SP of serous ovarian cancer cells. We transfected a library of 81,000 shRNA lentiviral plasmids (Cellecta) targeting 15,000 genes into two serous ovarian cancer cell lines, CH1 and SKOV3, which harbor minimal SP fractions (less than 0.1%). Following transfection, we sorted cells to obtain the SP fraction, expanded these from independently plated single SP cells and extracted DNA. We amplified the transduced shRNAs by PCR and reconstructed shRNA-lentiviral plasmids, followed by transfection into CH1 or SKOV3 to determine reproducibility. Furthermore, we transfected different sequences of shRNAs targeting the identified genes and confirmed the consequent increase in the SP fraction. As a result, suppression of GeneA, GeneB and GeneC markedly increased the SP in CH1 cells (control; 0.08% vs GeneA; 1.1%, GeneB; 1.0%, GeneC; 2.0%). Suppression of GeneD, GeneE and GeneF markedly increased the SP of SKOV3 cells (control; 0.07% vs GeneD; 1.4%, GeneE; 1.1%, GeneF; 1.0%). Moreover, we overexpressed five genes (GeneA, GeneC, GeneD, GeneE and GeneF) in two serous ovarian cancer cell lines, A2780 and IGROV1, which contain more abundant SP fractions and found a marked decrease of the SP fraction (IGROV1: control, 6.8% vs 0.65%, 2.1%, 4.2%, 0.34%, 0.35%, p<0.001, respectively, A2780: control, 3.48% vs 0.01%, 0.15%, 0.22%, 0.08%, 0.21%, p<0.001, respectively). We next analyzed phenotypic characteristics of CH1 cells in which GeneA, GeneB or GeneC is suppressed. In clonogenicity assays of single cells, we found that the SP had higher clonogenicity than the main population (MP) and control cells (control shRNA; 1%, sh-GeneA-SP vs. MP; 10.7% vs. 5.5%, sh-GeneB-SP vs MP; 8.5% vs. 2.2%, sh-GeneC-SP 3.0% vs. 1.0%, p<0.05, respectively). The CH1 SP had higher tumorigenicity than the MP following subcutaneous injection of 1,000 cells into NOD/SCID mice (control shRNA, 1/4; sh-GeneA-SP vs. MP, 2/4 vs. 1/4; sh-GeneB-SP vs MP, 4/4 vs. 1/4; sh-GeneC-SP vs MP, 4/4 vs. 0/4, respectively). Suppression of these genes significantly elevated cisplatin IC50 values of CH1 cells as compared to the control-control, 40.9nM; sh-GeneA, 110nM; sh-GeneB, 102.3nM; sh-GeneC, 120.3nM; p<0.05, respectively. In addition, suppression of GeneB contributed to paclitaxel resistance while suppression of GeneC enhanced resistance to gemcitabine and paclitaxel. (p<0.05). Also we investigated characteristics of SKOV3 cells in which GeneD, GeneE or GeneF was suppressed. In clonogenicity assays of single cells, we found that SP cells had higher clonogenicity than MP and control cells (p<0.05, respectively). Suppression of these three genes significantly increased the IC50 values of SKOV3 for liposomal doxorubicin, gemcitabine and paclitaxel (p<0.05, respectively). Suppression of GeneD significantly increased cisplatin IC50 values compared to control cells (p<0.05). In conclusion, we identified six genes, whose down-regulation contributed to an increase in the SP of serous ovarian cancer, through functional genomics screening. Moreover, suppression of these genes was associated with CSC phenotypes, including clonogenicity, chemoresistance and in vivo tumorigenicity. These results are important with regard to revealing the molecular mechanisms of tumor recurrence. (Gene names are masked owing to potential conflict with a patent.) Citation Format: Noriomi Matsumura, Koji Yamanoi, Susan K. Murphy, Junzo Hamanishi, Kaoru Abiko, Ken Yamaguchi, Tsukasa Baba, Masafumi Koshiyama, Ikuo Konishi. Identification through functional genomics screening of factors whose downregulation enhances the side population in ovarian cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr A38.

Interference with endogenous EZH2 reverses the chemotherapy drug resistance in cervical cancer cells partly by up-regulating Dicer expression.

Cervical cancer is one of the most common female malignancies in the world, and chemotherapeutic drug resistance is a major obstacle to cancer therapy. Enhancer of zeste homolog 2 (EZH2) is an enzymatic subunit of polycomb repressive complex 2 (PRC2) and catalyzes the repressive histone H3 lysine 27 trimethylation (H3K27me3). However, the role of EZH2 on the chemotherapy drug resistance in cervical cancers remains unclear. In the present study, the cervical carcinoma specimens and paired normal tissue specimens were obtained and the expression of EZH2 was detected by western blotting. The results showed that high levels of EZH2 were detected in cervical carcinoma tissues, compared with paired control tissues (**p < 0.01). Next, three pairs of shRNA specific to EZH2 were designed and used to interfere with endogenous EZH2 expression. Cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays following treatment with various concentrations of cisplatin in HeLa and HeLa/DDP cells. The MTT assay results showed that knockdown of EZH2 in HeLa/DDP cells caused a 2.29- or 1.83-fold decrease in the cisplatin IC50 values (for shRNA1-EZH2, 34.88 vs. 15.21μg/mL; p < 0.01; for shRNA3-EZH2, 34.88 vs. 19.09μg/mL; p < 0.01). The EZH2 activity was also suppressed by 3-deazaneplanocin A (DZNep), EZH2 inhibitor, and the results demonstrated that, meanwhile, DZNep potently inhibited cell viability of HeLa/DDP cells, partly by suppression the levels of EZH2 and H3K27me3, but not H3K27me2, which was detected by western blotting analysis. Moreover, cell migration assay results showed that knockdown of EZH2 decreased cell metastasis of cervical cancer cells. Furthermore, cell cycle was detected by fluorescence-activated cell sorting (FACS) assay and the results demonstrated that interference with EZH2 expression increased the percentage of cells at G0/G1 phase and the HeLa/DDP cells were blocked at G0/G1 phase. Interestingly, western blotting results revealed that higher expression of EZH2 was related with lower level of Dicer in HeLa/DDP cells. Finally, in vivo tumorigenicity experiments results demonstrated that interference with endogenous EZH2 by shRNA specific to EZH2 or inhibition EZH2 by DZNep could significantly increase antitumor effects in nude mice. Thus, inhibiting the levels of endogenous EZH2 effectively reversed the cisplatin resistance and increased the cisplatin sensitivity in cisplatin-resistant HeLa/DDP cells. EZH2 might be a potential target for treating chemotherapeutic drug-resistant cervical cancers.

Abstract 1639: Strong synergy with APR-246 and DNA-damaging drugs in primary ovarian cancer cells

Abstract Background: Mutations in the TP53 gene occur in at least 60% of ovarian tumors and are associated with chemoresistance and poor prognosis. APR-246 (PRIMA-1MET) is the first mutant p53-reactivating compound in clinical development and has been tested as monotherapy in hematological malignancies and prostate cancer with promising results (Lehmann et al. J Clin Oncol 30, 2012). The aim of this study was to investigate the anticancer effects of APR-246 in combination with conventional chemotherapy in cancer cells isolated from ascites fluid from ovarian cancer patients. Methods: Ascites cells were purified by Ficoll and viably frozen, and the quality and purity were confirmed by May Grünwald/Giemsa staining. For some samples, immunocytochemical stainings with anti-Ber-EP4 and anti-calretinin antibodies were used to distinguish between mesothelial and cancer cells. Cell viability was assessed with FMCA assay and Combination Index (CI) calculated using Additive model. CI &amp;lt; 0.8 indicates synergy and CI &amp;lt; 0.5 strong synergy. TP53 gene status was determined by Sanger sequencing and single strand conformation analysis, and p53 protein expression by Western blotting. Results: Eight of ten samples tested were from patients with recurrent ovarian cancer previously treated with platinum drugs. Cancer cells from seven patients possessed TP53 core domain missense mutations L111Q, C135Y, P151H, Y163H, C238F, P278R and R280K, respectively; two had nonsense mutations E346* and E204*, and one was wild type. All the missense mutations have been predicted to severely affect p53 tumor suppressor function. Missense mutant p53 proteins were expressed at high levels while no p53 expression was detected in cells with wild type or nonsense mutant p53. Synergistic or strong synergistic effects with APR-246 and cisplatin were observed in all ten samples tested. Synergy was also observed with the platinum analogue carboplatin and the anthracycline doxorubicin. The IC50 values for cisplatin ranged from 3 to 40 μM and for APR-246 from 5 to 37 μM. We also tested the ability of APR-246 to sensitize the primary ovarian cancer cells carrying Y163H mutant p53, to cisplatin; the IC50-value of cisplatin decreased from 10 to 2.6 μM in the presence of 6 μM APR-246. Conclusions: We observed striking synergy with APR-246 and platinum drugs or doxorubicin. These results are consistent with our previous results in ovarian cancer cell lines showing synergy not only in p53 mutant but also in p53 null cancer cell lines. In these cells, the synergy may be related to the fact that APR-246 decreases intracellular glutathione level. Our results provide a strong rationale for the ongoing clinical study with APR-246 in combination with carboplatin and doxorubicin in patients with recurrent ovarian cancer and suggest that combination treatment with APR-246 and DNA-damaging drugs could allow significantly improved treatment for ovarian cancer carrying mutant p53. Citation Format: Åsa Fransson, Daria Glaessgen, Jessica Alfredsson, Klas G. Wiman, Svetlana Bajalica Lagercrantz, Nina Mohell. Strong synergy with APR-246 and DNA-damaging drugs in primary ovarian cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1639. doi:10.1158/1538-7445.AM2015-1639

Abstract 585: Elevated levels of CD24 in head and neck squamous carcinoma cells: A potential marker for unfavorable cisplatin response

Abstract Introduction: Head and neck cancers are usually detected in their later stages of progression, resulting in a 5-year survival rate of only 30-50%. Chemotherapy is a treatment of choice for locally advanced head and neck squamous cellular carcinoma (HNSCC), and unfortunately, only a subset of HNSCC responds favorably to cisplatin treatment. Currently, there are no predictive markers of a tumor's response to cisplatin therapy. Therefore, to investigate the extremely low rates of durable remission in HNSCC, we employed a method of screening membrane bound surface stem cell markers as a means to possibly identify a group of cells that are inherently resistant to high doses of cisplatin treatment. Methods: Flow cytometry, MTT assay, QPCR, CD24 RNA interference, western blot, orosphere formation assay and IHC Results: Three UM-SCC cell lines were taken that had variable degrees of sensitivity to cisplatin, but all the lines formed a pronounced cisplatin resistant tail in a dose response curve. In an effort to screen for the fraction of cells that formed the cisplatin resistant tail, we observed that these three UM-SCC lines that expressed similar amounts of membrane bound CD44 had variable levels of membrane bound CD24 initially; however, all showed higher levels of CD24 with increased cisplatin dosage. Levels of CD24 peaked at the super high doses of cisplatin that had resulted in a cisplatin resistant tail. To further our study on the relationship between CD24 and cisplatin resistance, UM-SCC-10B and UM-SCC-10B/pt15s, a cisplatin sensitive-resistant pair from the larynx that express high amounts of CD24 in normal conditions, were selected. Upon confirmation of CD24 up-regulation at a clinically relevant dose of cisplatin [10uM], a lentiviral vector construct containing a CD24 siRNA was introduced into the model pair. The knockdown of CD24 resulted in increased sensitivity to cisplatin, and it diminished the cisplatin resistant tail in a dose response curve. Also, CD24 knockdown lines had significantly lower cisplatin IC-50 values. Upon treating the CD24 high UM-SCC-10B and UM-SCC-10B/pt15s with a combination of monoclonal CD24 antibody and cisplatin, higher cisplatin sensitivity was achieved. This was followed by screening of CD24 expression in head and neck tumor tissue samples from patients that had undergone cisplatin treatment. The tissue screening further confirmed the correlation between CD24 overexpression and unfavorable cisplatin treatment response. Conclusion: Our work thus far, indicates that higher expression of CD24 in head and neck tumors result in a cisplatin resistant population that may well be the cause of unfavorable response to cisplatin treatment. Overall, CD24 has the potential to be a valuable predictor of response to cisplatin in HNSCC patients as well as a therapeutic target. Citation Format: Vishnu Modur. Elevated levels of CD24 in head and neck squamous carcinoma cells: A potential marker for unfavorable cisplatin response. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 585. doi:10.1158/1538-7445.AM2015-585

Interference with HMGB1 increases the sensitivity to chemotherapy drugs by inhibiting HMGB1-mediated cell autophagy and inducing cell apoptosis.

Non-small cell lung cancer is commonly seen with higher morbidity and mortality. High-mobility group protein 1 (HMGB1) is a highly conserved nuclear protein, which is involved in multiple human diseases including cancers. However, the mechanisms of HMGB1 in non-small cell lung cancer remain unclear. The goal of the present study is to identify the relationship between HMGB1 and the progresssion of non-small cell lung cancer and investigate the molecular mechanism of HMGB1 in non-small lung cancer cell lines. Firstly, we detected the expression levels of HMGB1 by by real-time PCR and western blotting analysis, and the results demonstrated that HMGB1 was much higher expressed in non-small cell lung cancer cell lines, including A549, SPC-1-1, NCI-2170, SK-MES-1, and NCI-H1299, compared with that of WI-38. Next, 5 μM of adriamycin (AMD), 20 μM of cisplatin (DDP), and 50 μM of methotrexate (MTX) were used to treat A549 cells and SPC-A-1 cells for 48 h. The results showed that treatment with chemotherapy drugs significantly increased the levels of HMGB1 in A549 cells and SPC-A-1 cells. Moreover, the expression levels of HMGB1 increased in a time-dependent manner being treated with DDP. Then, the endogenous HMGB1 expression was successfully interferred with shRNA specific to HMGB1 in A549 and SPC-A-1 cells, which was detected by western blotting analysis. Then, the cisplatin-sensitive A549 cells and cisplatin-resistant A549/DDP cells were treated with increasing concentrations of cisplatin for 24, 48, and 72 h; cell viability were analyzed by MTT assay; and IC50 values were calculated. The results demonstrated that the expression level of HMGB1 in A549/DDP cells was much higher than that of A549 cells; moreover, transfection with HMGB1 shRNA in A549/DDP cells decreased the IC50 value of cisplatin in A549/DDP cells. The expression levels of autophagy-related proteins beclin-1 and LC3-II were significantly higher in A549/DDP cells or the A549 cells treated with chemotherapeutic drugs, compared with that in A549 cells. However, interference with endogenous HMGB1 obviously suppressed autophagy-related proteins and increased cell apoptosis rate and the expression of cleaved caspase-3 in A549/DDP cells. All of the data suggested that interference with the endogenous HMGB1 significantly inhibited cell autophagy and increased cell apoptosis of A549/DDP cells. Thus, the study on the resistance of chemotherapy drugs would provide a theoretical reference for clinical treatment of non-small cell lung cancer.

Twist promotes invasion and cisplatin resistance in pancreatic cancer cells through growth differentiation factor 15.

Pancreatic cancer (PC) is an aggressive and devastating disease with a poor prognosis. Cisplatin, a commonly used chemotherapeutic agent for solid tumors, is effective as a single agent or in combination with other drugs for the treatment of PC. Previous studies have suggested that Twist and growth differentiation factor 15 (GDF15) are involved in the progression of PC. However, the role of Twist and GDF15 in PC remains to be elucidated. In the present study, the individual effect of and interaction between Twist and GDF15 in PC cell invasion and chemoresistance to cisplatin was examined. Twist and/or GDF15 were stably overexpressed or knocked down in ASPC‑1 and BXPC‑3 human PC cells. Overexpression of Twist in the two cell lines markedly increased GDF15 expression, cell invasion, matrix metalloproteinase‑2 expression/activity and the half maximal inhibitory concentration (IC50) values of cisplatin, which was eradicated by GDF15 knockdown or the selective p38 mitogen‑activated protein kinase (MAPK) inhibitor SB203580 (10 µM). By contrast, Twist knockdown significantly decreased GDF15 expression, cell invasion, matrix metalloproteinase‑2 expression/activity and the IC50 values of cisplatin, which was completely reversed by overexpression of GDF15. In addition, while overexpression and knockdown of Twist increased and decreased p38 MAPK activity, respectively, GDF15 demonstrated no significant effect on p38 MAPK activity in PC cells. In conclusion, the present study, for the first time, to the best of our knowledge, demonstrated that Twist promotes PC cell invasion and cisplatin chemoresistance through inducing GDF15 expression via a p38 MAPK‑dependent mechanism. The present study provides new insights into the molecular mechanisms underlying PC progression and chemoresistance.

Predicting chemosensitivity to gemcitabine and cisplatin based on gene polymorphisms and mRNA expression in non-small-cell lung cancer cells.

We used a panel of 17 non-small-cell lung cancer cell lines to investigate whether the presence of polymorphisms in the RRM1, ERCC1, ABCB1 and MTHFR genes and alterations in their mRNA expression can affect the in vitro chemosensitivity to cisplatin and gemcitabine. Polymorphisms in these genes were evaluated by direct sequencing. mRNA expression levels were assessed by realtime PCR. In vitro chemosensitivity to cisplatin and gemcitabine was expressed as IC50 values, using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay. There was a significant, positive correlation between RRM1 mRNA expression and IC50 values for gemcitabine (r = 0.6533, p = 0.0045), and there was a significant, negative correlation between ABCB1 mRNA expression and IC50 values for cisplatin (r = -0.5459, p = 0.0287). When examining the association between the polymorphisms and IC50, we found that only the MTHFR 1298A>C polymorphism showed a tendency to be more chemosensitive to gemcitabine (p = 0.0440). These in vitro results suggest that mRNA expression levels of the RRM1 and ABCB1 genes may be useful indicators of chemosensitivity to gemcitabine and cisplatin, respectively. The MTHFR 1298A>C polymorphism was associated with gemcitabine chemosensitivity, which require further functional analysis with co-expressed genes and should be explored in prospective clinical studies to determine its potential clinical application as a predictive biomarker. Original submitted 11 February 2014; Revision submitted 3 November 2014.

Dynamic monitoring of the cytotoxic effects of protoberberine alkaloids from Rhizoma Coptidis on HepG2 cells using the xCELLigence system

AimTo investigate the cytotoxic effects of the six protoberberine alkaloids (PAs) from Rhizoma Coptidis on HepG2 cells. MethodA systematic screening was conducted to investigate the dynamic response of HepG2 cells to the PAs using the impedance-based xCELLigence system. Cisplatin was selected as the positive control. The real time, concentration-response curves and the 50% inhibitory concentrations (IC50) were acquired to evaluate the anticancer activity of the PAs. ResultsAll of the six PAs inhibited cell growth and induce death in HepG2 cells in a time- and concentration-dependent manner. The IC50 values of cisplatin, berberine, columbamine, coptisine, epiberberine, jatrorrhizine, and palmatine were 5.13, 42.33, 226.54, 36.90, 302.72, 383.54, and 456.96 μ·mL−1, respectively. The results obtained using the xCELLigence system corresponded well with those of the conventional methods. ConclusionThe xCELLigence system is a reliable and efficient tool for real-time screening of the cytotoxic effect of compounds in cell-based in vitro assays. Coptisine and berberine, with methylenedioxy group at C2 and C3 on the phenyl ring showed stronger effect.than the other four PAs. However, compared with cisplatin, the six PAs didn't show obvious cytotoxic effect on HepG2 cells. These results provided some useful data for the evaluation of the anticancer compounds, and the clinical application of traditional Chinese medicine.

Chemosensitivity profile of HCT-116 colorectal cancer cells in 3D spheroids compared to 2D monolayers.

e14606 Background: Personalizing cancer chemotherapy using ex-vivo cytometric profiling assays of live cancer cells hold great promise to significantly improve the patient's chances for tumor response and survival (Apfel et al. ASCO 2013, e22188). However, most assays dissociate the tumor into single cells and measure the response to the chemotherapy on a 2-dimensional (2D) monolayer, but there is concern that tumor response to chemotherapy in its native 3-dimensional (3D) state may be different. Methods: We exposed 3200 HTC-116 colon cancer cells either as monolayers or as spherioids of about 180 microns diameter to 0, 0.01, 0.3, 1, 3, 10, 30 and 100 times the commonly accepted IC50 values of cisplatin, topopecan, 5-FU, and tirapazamine. After 72 hours exposure, viable cell numbers were determined by measuring ATP using luciferin-luciferase chemiluminescence. We then determined IC50s using GraphPad Prism and the respective ratios. Results: IC50s of spheroids vs. 2D monolayers were 10 vs. 4.8 micromol/L for cisplatin, 18 vs. 11 nmol/L for topotecan, 4.8 vs. 4.3 micromol/L for 5-FU, and 65 vs. 130 micromol/L for tirapazamine, respectively. The ratios (95% CI) of the EC50s were 2.13 (1.03-4.39) for cisplatin, 1.61 (0.93-2.81) for topotecan, 1.1 (0.63-1.94) for 5-FU, and 0.49 (0.29-0.81) for tirapazamine. Conclusions: The EC50 for 3D spheroids compared with 2D monolayers is higher for cisplatin, the same for topotecan and 5-FU, and lower for tirapazamine. Chemosensitivity results from a 3D tumor model may be more realistic than results from 2D monolayer assays.

Epstein-Barr Virus_Encoded LMP1 Upregulates MicroRNA-21 to Promote the Resistance of Nasopharyngeal Carcinoma Cells to Cisplatin-Induced Apoptosis by Suppressing PDCD4 and Fas-L

Approximately 30% of patients with Epstein-Barr virus (EBV)-positive advanced nasopharyngeal carcinoma (NPC) display chemoresistance to cisplatin-based regimens, but the underlying mechanisms are unclear. The Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP1), a functional homologue of the tumor necrosis factor receptor family, contributes substantially to the oncogenic potential of EBV through the activation of multiple signaling pathways, and it is closely associated with a poorer prognosis for NPC. Recent studies show that EBV infection can induce the expression of many cellular miRNAs, including microRNA-21, a biomarker for chemoresistance. However, neither a link between LMP1 expression and miR-21 upregulation nor their cross talk in affecting chemoresistance to cisplatin have been reported. Here, we observed that stable LMP1-transformed NPC cells were less sensitive to cisplatin treatment based on their proliferation, colony formation, the IC50 value of cisplatin and the apoptosis index. Higher levels of miR-21 were found in EBV-carrying and LMP1-positive cell lines, suggesting that LMP1 may be linked to miR-21 upregulation. These data were confirmed by our results that exogenous LMP1 increased miR-21 in both transiently and stably LMP1-transfected cells, and the knock down of miR-21 substantially reversed the resistance of the NPC cells to cisplatin treatment. Moreover, the proapoptotic factors programmed cell death 4 (PDCD4) and Fas ligand (Fas-L), which were negatively regulated by miR-21, were found to play an important role in the program of LMP1-dependent cisplatin resistance. Finally, we demonstrated that LMP1 induced miR-21 expression primarily by modulating the PI3K/AKT/FOXO3a signaling pathway. Taken together, we revealed for the first time that viral LMP1 triggers the PI3K/Akt/FOXO3a pathway to induce human miR-21 expression, which subsequently decreases the expression of PDCD4 and Fas-L, and results in chemoresistance in NPC cells.

Inhibition of FOXM1 by thiostrepton sensitizes medulloblastoma to the effects of chemotherapy

Medulloblastoma (MB) is the most common malignant brain tumor in children and is highly invasive and metastatic. Despite recent advances, most MB patients suffer significant therapy-related morbidity, and the survival rate for patients with metastatic MB remains unsatisfactory. Altered expression of FOXM1 has been detected in many types of cancers, and the inhibition of FOXM1 has been studied as a cancer therapy. In the present study, we evaluated the impact of the inhibition of FOXM1 by thiostrepton in Daoy MB cells. Cells were treated with different concentrations of thiostrepton alone or in combination with cisplatin. Cell viability was measured with CCK-8 assays, and cell cycle distribution and apoptosis were assessed by flow cytometric analysis. Changes in protein expression were examined by western blotting. RNAi experiments were performed using siRNA oligonucleotides. The invasion and migration studies were performed using 8-µm Transwell plates. Inhibition of FOXM1 by thiostrepton significantly decreased MB cell proliferation. Cell arrest at the G2/M phase and apoptosis were significantly increased in MB cell lines that were treated with thiostrepton or transfected with siRNA. Thiostrepton decreased the IC50 value of cisplatin for MB treatment by enhancing cisplatin-induced apoptosis. Thiostrepton also decreased cell invasion and migration, which are crucial steps for tumor progression. Our data suggest that targeting FOXM1 with small-molecule inhibitors results in potent antitumor activity and chemosensitizing effects in human medulloblastoma cells.

Effect of bortezomib in combination with cisplatin and 5-fluorouracil on 4T1 breast cancer cells

Bortezomib is a highly selective and reversible inhibitor of the 26Sproteasome. It has been approved for the treatment of patients with relapsed and refractory multiple myeloma. A number of studies have been conducted to evaluate the activity and safety of bortezomib either alone or in combination with several cytotoxic agents and radiation. In the current study, the efficacy of bortezomib alone or in combination with cisplatin and 5‑fluorouracil was evaluated in 4T1breast cancer cells, a highly metastatic murine cancer cell line. Using MTT assay, IC50values of cisplatin and 5‑fluorouracil were determined to be 14.2and 8.9µM for cisplatin and 5‑fluorouracil, respectively. The effects of different concentrations of cisplatin and 5‑fluorouracil in combination with two different concentrations of bortezomib were examined in the 4T1cells. Statistically significant differences were found when 1 or 5µM cisplatin was combined with 10 or 50nM bortezomib. Similarly, 1µM 5‑fluorouracil or 5µM 5‑fluorouracil in combination with 10nM bortezomib caused significant cell death as compared to treatment with single agents. However, 1or 5µM 5‑fluorouracil did not potentiate the effects of higher concentrations of bortezomib (50nM). The effect of the combination of cisplatin, 5‑fluorouracil and bortezomib was determined by soft agar assay. It was confirmed that a combination of cisplatin and bortezomib was more effective than each drug as a monotherapy. Therefore, the combination of cisplatin and bortezomib should be tested further in clinical settings.

Abstract 232: Cancer stem cells and cisplatin.

Abstract Introduction: Cisplatin is an important chemotherapeutic agent, which has resulted in higher overall response rates when combined with 5-FU or taxane combinations in treating squamous cellular carcinoma of the head and neck. However, HNSCC tumor response rates to cisplatin range from 20-30%, a major clinical hurdle today. To address this issue thoroughly, we investigated the resistance to cisplatin in HNSCC under the purview of the cancer stem cell hypothesis, using the widely studied CSC marker combinations of, CD44+ 24- and ALDH-Hi. In order to get a broader perspective on this issue, we worked on multiple HNSCC tumor cell lines with varying degrees of cisplatin resistance, arising from tumors of the pharynx, larynx, tongue, tonsils, alveolus, hypopharynx and supraglottis. Methods: Seven HNSCC cell lines were analyzed for their CSC percentage through FACS, utilizing CD44+ 24- and ALDH-Hi marker systems. Cisplatin IC-50 for each of these seven cell lines was then determined by plotting percent viability vs. drug concentration curves. The remaining viable cells after cisplatin treatment were then analyzed through FACS for their CD44+ 24- and ALDH-Hi percentages in order to compare with the CSC percentage in the untreated HNSCC lines. Results: There was a wide variation in the percentages of CD44+24- and ALDH-Hi cells across these seven HNSCC lines. An inverse correlation between the initial percentages of CD44+24- and ALDH-Hi cells and the corresponding cisplatin IC-50 values was seen. HNSCC cell lines that had lower IC-50 values for cisplatin displayed greater cell viability at higher concentrations of cisplatin. This was evident by a long tail in the sigmoidal dose response curve. The number of viable cells at the end of cisplatin treatment had a linear relationship with the overall percentage of CSCs in the control untreated cell lines. Conclusion: Our results suggest that the resistance offered by the CD44+24- and ALDH1-Hi cells is apparent only once the initial 50% loss of cells occurs. This in vitro biphasic decline in percent viable cells clearly indicates that HNSCC cisplatin resistance is a twofold problem. The bulk of non-cancer stem cells within a tumor can acquire resistance to cisplatin via multiple molecular pathways, but this is only a front line cover of resistance to the resistance offered by the cancer stem cells. Citation Format: Vishnu Modur. Cancer stem cells and cisplatin. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 232. doi:10.1158/1538-7445.AM2013-232

Abstract 2488: Novel candidate protein biomarkers for cisplatin response prediction in non-small cell lung cancer.

Abstract The five-year survival rate for non-small cell lung cancer (NSCLC) is still less than twenty percent in part due to treatment failure and lack of biomarkers for personalized therapy. Most NSCLC patients are treated by chemotherapy and cisplatin is still the major component of most chemotherapy regimens. However, resistance to cisplatin is a common phenomenon. Aim of this study was to identify protein biomarkers that can be used for cisplatin response prediction. The proteomes and secretomes1 of a series of human NSCLC cell lines with a range of IC50-values for cisplatin (1.5 - 15 μM) were analyzed by label-free proteomics based on GeLC-MS/MS and quantified by spectral counting. Significant differential expression of proteins was determined by the in-house developed beta-binomial statistical test2. Network and gene ontology analysis was performed, with STRING and DAVID respectively, to reveal cellular and molecular functions associated with the differential proteins. In total, 2885 and 2342 proteins were detected in the cell lysates and secretomes, respectively. The proteins with significantly higher expression in the resistant cell lines were associated RNA processing and vesicle transport, while the proteins with higher expression in the sensitive cell lines were known to be involved in RNA splicing, DNA damage repair and extracellular matrix organization. The most promising candidates are being validated in tissues and sputum of patients with known response to cisplatin. 1 Piersma SR et al. J Proteome Res. 2010 Apr 5;9(4):1913-22. 2 Pham TV et al. Bioinformatics. 2010 Feb 1;26(3):363-9. Citation Format: Tieneke B M Schaaij-Visser, Remco Nagel, Inge de Reus, Sander R. Piersma, Thang V. Pham, Egbert F. Smit, Frederik B. Thunnissen, Ruud H. Brakenhoff, Anton Berns, Connie R. Jimenez. Novel candidate protein biomarkers for cisplatin response prediction in non-small cell lung cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2488. doi:10.1158/1538-7445.AM2013-2488

Establishment and characterization of a novel ovarian clear cell adenocarcinoma cell line, TU-OC-1, with a mutation in the PIK3CA gene

A new cell line of human ovarian clear cell adenocarcinoma (CCC), TU-OC-1, was established and characterized. The cells showed a polygonal-shaped morphology and grew in monolayers without contact inhibition and were arranged like a jigsaw puzzle. The chromosome numbers ranged from 64 to 90. A low rate of proliferation was observed, similar to other CCC cell lines tested (OVTOKO, RMG-I, and OVAS), and the doubling time was 38.4h. The respective IC50 values of cisplatin and paclitaxel were 12.2μM and 58.3nM. Mutational analysis revealed that TU-OC-1 cells harbored a PIK3CA mutation at codon 542 (E542K) in exon 9, which is a mutation hot spot on this gene. We observed that phosphorylated Akt protein was overexpressed in TU-OC-1 cells by western blot analysis. Heterotransplantation to nude mice produced tumors that reflected the original. This cell line may be useful to study the chemoresistant mechanisms of CCC and contribute to novel treatment strategies.

Overexpression of IL-7 enhances cisplatin resistance in glioma

Cisplatin is one of the most commonly used chemotherapeutic agents for glioma patients. In this study, array comparative genomic hybridization (aCGH) was used to identify genes associated with cisplatin resistance in a human glioma cell line. The cisplatin-resistant U251/CP2 cell line was derived by stepwise selection using cisplatin. The genetic aberrations of the U251 parental cell line and the U251/CP2 cells were analyzed using aCGH. RT-PCR was used to detect the expression of the altered genes revealed by aCGH. The sensitivity of glioma cells to cisplatin was determined by using the MTT assay. Apoptosis was detected using flow cytometry and western blot analysis. The IC50 value of cisplatin in U251/CP2 cells was five times higher than its IC50 in U251 cells. The U251 cells lost at least one copy each of the CFHR1 and CFHR3 genes, and both CFHR1 and CFHR3 were homozygously deleted in U251/CP2 cells. The U251/CP2 cells gained two to three copies of C8orf70 and IL-7 genes. IL-7 mRNA expression was studied in 12 glioma cell lines, and expression was positively correlated with the IC50 of cisplatin. Furthermore, IL-7 mRNA expression was also positively correlated with the IC50 of cisplatin in 91 clinical glioma specimens. Additionally, treatment with recombinant human IL-7 (rhIL-7) enhanced cisplatin resistance and increased the relative growth rate of the glioma cells. Moreover, the apoptosis induced by cisplatin could be inhibited by IL-7. In conclusion, our results suggest that IL-7 may play an important role in cisplatin resistance in glioma.

Abstract 2140: Regulator of G-protein signaling 2 blunts cellular viability mediated by a stabilized lysophosphatidic acid analogue (2S-OMPT) in the presence of chemotherapy in an ovarian cancer cell model of chemoresistance

Abstract Lysophosphatidic acid (LPA) enhances growth, survival, viability and proliferation in numerous models. Thus, the receptors for LPA are also implicated in oncogenic signaling and represent a group of “druggable” therapeutic targets in cancer. Regulator of G protein Signaling 2 (RGS2) is a GTPase activating protein (GAP) that deactivates the G alpha subunits of heterotrimeric G proteins, turning off G protein-coupled receptor (GPCR) signaling. In our previous studies examining computational bioinformatics, we demonstrated that reduced RGS gene expression develops with the occurrence of drug resistance in ovarian cancer cell lines. The purpose of our study was to investigate whether the LPA receptors are also involved in mediating viability as a result of enhanced GPCR signaling due to reduced RGS proteins in this chemoresistance model. This is important since RGS proteins can deactivate G alpha subunits of many different GPCRs, and are not specific to merely the LPA receptors. If a “druggable” GPCR is mediating viability in chemoresistance, it is critical to identify which ones are responsible. To test this idea we used the metabolically stabilized LPA analogue L-sn-1-O-oleoyl-2-O-methylglyceryl-3-phosphothionate (2S-OMPT) as a potent non-hydrolyzable agonist for LPA receptors at a concentration (10 µM) sufficient to activate multiple LPA receptors. We hypothesized that if LPA signaling was not involved, then OMPT would override any contribution of RGS2 on modulating the viability response in the presence of chemotherapy. Using a tet-off promoter system, we generated an inducible stable model of RGS2-expressing HeyA8-MDR cells, which we have previously confirmed are resistant to taxanes. After 48 h OMPT pre-treatment, HeyA8-MDR-RGS2-inducible cells were exposed to cisplatin for 24 h and evaluated for viability. IC50 values for cisplatin were lower in the RGS2-induced HeyA8-MDR cells that were not pretreated with OMPT, a demonstration of enhanced sensitivity to cisplatin in the presence of RGS2. All conditions pre-treated with OMPT demonstrated enhanced viability even in the presence of chemotherapy; however, ovarian cancer cells that were pretreated with OMPT and cultured in tet-free medium to induce RGS2 expression showed a significant blunting of viability. This supports the interaction between LPA receptors and RGS2 in the control of drug sensitivity in ovarian cancer chemoresistance and provides a mechanism for our current and previous observations. 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 2140. doi:1538-7445.AM2012-2140

Treatment of small cell lung cancer with TRA-8 in combination with cisplatin and radiation

BackgroundLimited stage small cell lung cancer (SCLC) represents a minority of SCLC. Despite extensive clinical trials, standard treatment remains cisplatin-based chemotherapy and thoracic irradiation (TI). This study focused on the interaction of cisplatin/radiation with the anti-human DR5 monoclonal antibody TRA-8 in SCLC cells. TRA-8 binds specifically to DR5 and has been shown to activate apoptosis. MethodsFour human SCLC cell lines were utilized for experimentation (SCLC-41, SCLC-58, SCLC-68, and SCLC-74). Immunoblot analysis was used to determine relative protein levels of DR5, DR4 and pro-caspase 8 for each cell line. Using a tetrazolium-based assay (XTT), the IC50 values for cisplatin with or without TRA-8 were determined for the SCLC cell lines. Four SCLC lines were assayed with a combination of TRA-8 (10μg/ml), 2Gy radiation and various concentrations of cisplatin. Apoptosis was evaluated using Annexin V-FITC and cleaved caspase immunoblotting. Using a SCLC-58 subcutaneous xenograft model, treatment began 21d after tumor cell injection. Treatment included weekly cisplatin (4mg/kg) and radiation of 1Gy (24h after cisplatin) and TRA-8 (200μg) was administered i.p. twice weekly for three weeks. ResultsImmunoblot analysis showed similar levels of DR5 for all cell lines with variable levels of DR4. Various concentrations of TRA-8 antibody (⩽10μg/ml) induced no significant cytotoxicity in the SCLC cell lines. The in vitro combination treatment with TRA-8 (10μg/ml), 1.25μg/ml cisplatin and 2Gy radiation showed increased cytotoxicity when compared to combinations without TRA-8. Furthermore, the triple combination demonstrated the greatest amount of apoptosis as measured by Annexin V staining. The in vivo studies showed the combination of 1Gy, cisplatin and TRA-8 extended the tumor doubling time to 44d as compared to any doublet treatment groups that ranged from 12 to 20d. Analysis of survival data showed 100% of the combination group (RT+cisplatin+TRA-8) were alive 65d after treatment began whereas all doublet treatment groups showed 50% or less survival. ConclusionsThese studies showed increased cytotoxicity when TRA-8 was added to radiation/cisplatin in SCLC. This effect was demonstrated in vitro and in vivo. TRA-8 represents a promising new agent in the treatment of SCLC.

The use of PARP inhibitors as single agents and as chemosensitizers in sporadic pancreatic cancer.

e13542 Background: Introduction: After resection of pancreatic cancer local recurrence occurs in 50%-80% of the cases while distant metastasis develops 75% of the time. Most pancreatic cancers have impairments of DNA repair. PARP inhibitors may further inhibit the ability fo the cancer to undergo DNA repair. Current, adjuvant therapy often consist of gemcitabine, cisplatin and/or 5-fluorouracil which add a modest increase in median survival by 4-5 months. In this study, we treated human pancreatic cancer cells with poly(ADP-ribose) polymerase (PARP) Inhibitors (AG14361, veliparib and olaparib) alone or with gemcitabine, cisplatin or 5-fluorouracil. Methods: CFPAC-1 and BXPC-3 , HPAC human pancreatic cancer cell lines were treated for 72 hours with PARP inhibitors alone or in combination with gemcitabine, cisplatin, or 5-fluorouracil. Validated MTT assays were used to form dose response curves from which the IC50 values were calculated. PARP activity was measured indirectly through PAR levels and correlated with the IC50 values of PARP inhibition of each cell line. Results: The PARP1 IC50 values for CFPAC-1, BXPC-3 and HPAC pancreatic cancer cell lines were AG14361 (14.3 µM, 12.7 µM, 38.3 µM ), veliparib (52.6 µM, 100.9 µM 102.0 µM ) and olaparib (79.5 µM, 184.8 µM, 200.2 µM) .These values indicate a sensitivity to PARP1 inhibitors of pancreatic cancer cell lines that is at least as great as the sensitivity of ovarian and breast cancer cell lines to PARP1 inhibition. The IC50 values of cisplatin, were decreased up to 60 fold in the presence of clinically relevant amounts of PARP inhibitor while 5-fluorouracil IC50 values were decreased up to 6000 fold in the presence of clinically relevant amounts of PARP inhibitor. Gemcitabine was inhibited up to 73% by PARP inhibitors. The was poor correlation between PARP activity and teh ability of PARP inhibitors to inhibit pancreatic cancer growth. Conclusions: Sporadic pancreatic cancer cells are exquisitely sensitive to PARP inhibition. PARP inhibitors significantly enhanced the cytotoxicity of cisplatin and 5-fluorouracil while inhibiting gemcitabine. There is little correlation between endogenous PARP activity and the effectiveness of PARP inhibitors to inhibit cancer growth.