Cisplatin In Lung Cancer Cells Research Articles

Confluence-dependent resistance to cisplatin in lung cancer cells is regulated by transforming growth factor-beta

ABSTRACTPurpose of the Study: Confluence-dependent resistance (CDR) is a phenomenon in which the efficacy of anti-cancer agents decreases when cell density increases. CDR in lung cancer has never been reported. The purpose of this study is to investigate if CDR can occur in NSCLC cells and to find a role for transforming growth factor (TGF)-β as a mechanism of CDR. Materials and Methods: Non–small cell lung cancer (NSCLC) cell lines A549 and H2228 were exposed to cisplatin in a variety of cell density conditions. RNA interference targeting TGF-β receptor I was performed to silence the TGF-β pathway. Results: CDR to cisplatin was induced in NSCLC cells, whereas CDR to crizotinib, an inhibitor of activin receptor-like kinase, was not observed. During confluent conditions, the TGF-β1 concentration in the culture medium was the highest. Exogenous TGF-β1 inhibited cell proliferation and reduced sensitivity to cisplatin. Inhibition of the TGF-β pathway increased in terms of sensitivity to cisplatin at confluency. Conclusions: CDR to cisplatin can occur in NSCLC cells, and the TGF-β pathway is associated with the regulation of CDR.

EGFR kinase domain mutation positive lung cancers are sensitive to intrapleural perfusion with hyperthermic chemotherapy (IPHC) complete treatment.

Lung cancer is the global leading cause of cancer-related deaths. A significant portion of lung cancer patients harbor kinase domain mutations in the epidermal growth factor receptor (EGFR). While EGFR tyrosine kinase inhibitors (TKI) effectively shrink tumors harboring mutant EGFR, clinical efficacy is limited by the development of TKI resistance. Effective alternatives are desperately needed in clinic for treating EGFR kinase domain mutation positive lung cancer. In our clinic in treating M1a lung cancer patients through intrapleural perfusion with hyperthermic chemotherapy (IPHC) followed by cycles of systemic chemotherapy (we termed this procedure IPHC complete treatment, IPHC-CT), we found dramatic tumor shrinkage in mutant EGFR-positive patients. We further confirmed the sensitivity of EGFR mutation-positive lung cancer cell lines derived from patients to HC (hyperthermic chemotherapy) treatment. We found that hyperthermia promoted accumulation of cisplatin in lung cancer cells. Hyperthermia and cisplatin synergistically downregulated the EGFR protein level, leading to quenching of signal from EGFR and induction of apoptosis. Our work therefore showed IPHC-CT is an effective treatment for EGFR kinase domain mutation positive lung cancer patients.

Abstract 2523: Strong synergistic effects with APR-246 and cisplatin in p53-mutant lung cancer cells

Abstract Background: Platinum compounds have been used as first-line treatment for many solid tumors including non small cell (NSCLC) and small cell (SCLC) lung cancer. However, patients with lung cancer often develop resistance to platinum compounds and eventually die of chemotherapy refractory disease. Mutation in the tumor suppressor protein p53 is common in lung cancer, ranging from 33% in adenocarcinomas to 70% in SCLC (The p53 website, http://p53.free.fr), and is one of the main causes for resistance to chemotherapy. APR-246 (PRIMA-1MET) is the first compound in clinical development that reactivates mutant p53 by inducing its wild type conformation thus triggering apoptosis (Lambert et al. Cancer Cell 15, 2009). APR-246 has yielded promising results in a first-in-human clinical trial in patients with hematological malignancies and prostate cancer (Lehmann et al. J Clin Oncol 30, 2012), and a Phase Ib/II study in combination with platinum-based therapy in ovarian cancer is ongoing. Previously we have shown strong synergy with APR-246 and platinum compounds in p53-mutant drug-resistant ovarian cancer cells (AACR abstract # 3448, 2013). Moreover, APR-246 completely restored the sensitivity of cisplatin to resistant p53-mutant ovarian cancer cells (AACR abstract #1801, 2014). The aim of the current study was to investigate whether strong synergistic effect can also be observed in p53-mutant lung cancer cells. Methods: Cell viability was determined with FMCA or Cell Titer-Glo assay, p53 gene status by Sanger sequencing and single strand conformation analysis, and p53 protein expression by Western blotting. Combination Index (CI) was calculated according to Additive model. Results: We observed strong synergistic effect (CI<0.5) with APR-246 and cisplatin in lung cancer cell lines carrying homozygous p53 hotspot mutations; NCI-H1770 (R248W), NCI-H1975 (R273H), NCI-H596 (G245C), PC-14 (R248W) and PC-14/CDDP (R248W). All these cell lines expressed a high level of p53. Synergistic (CI<0.8) or strong synergistic effect was found in lung cancer cell lines NCI-H378 (Y163C) and NCI-2087 (V157F) with homozygous mutations that occur frequently but are not hotspot mutations. NCI-2087 cells with the smoking-related V157F mutation express a high level of p53, while NCI-H378 cells express lower level. Mixed antagonist/additive/synergistic effects were observed in the p53 null cell line HOP-62, which does not express p53. Conclusions: Treatment with APR-246 in combination with cisplatin resulted in strong synergistic effect in both NSCL and SCLC cancer cells. Strongest synergies were observed in lung cancer cells with p53 hotspot mutations expressing a high level of p53. Studies to investigate the molecular mechanisms underlying the synergistic effects are ongoing. Our results suggest that combination treatment with APR-246 and platinum drugs may allow an improved therapy of p53-mutant lung cancer. Citation Format: Nina Mohell, Åsa Fransson, Jessica Alfredsson, Mikael von Euler, Ulf Björklund, Lars Abrahmsen. Strong synergistic effects with APR-246 and cisplatin in p53-mutant lung 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 2523. doi:10.1158/1538-7445.AM2015-2523

Abstract 5074: Activation of TGF β-IL6 axis mediates chemoresistance in non-small cell lung cancer by increasing epithelial-mesenchymal transition signaling

Abstract The tumor microenvironment is composed of different types of stromal cells that represent a key component of tumor progression, while it has been speculated that stromal components are functionally organized to promote cancer cell survival. A specific subset of stromal cells, termed cancer-associated fibroblasts (CAFs), secretes factors that promote tumorigenesis. We previously reported that CAFs increase the malignant potential of non-small-cell lung cancer (NSCLC) via the TGF β-IL6 axis. The purpose of this study was to clarify the role of the TGF β-IL6 axis in communication between CAFs and NSCLC cells for chemoresistance. Methods We used standard NSCLC cell lines (A549, NCI-H358), NSCLC cells obtained using a cancer tissue-originated spheroid (CTOS) method, lung normal fibroblasts (LNFs), and CAFs obtained from primary cultures of specimens from NSCLC patients to evaluate phenotypic changes in the presence of human IL-6, TGF-β1, and conditioned medium (CM) from the cells. Immunohistochemistry was also utilized to examine the distribution of fibroblasts in 75 tumor specimens obtained from patients with NSCLC after undergoing chemoradiotherapy. Results Expression levels of myofibroblast markers were elevated in CAFs as compared to LNFs, while CM from fibroblasts induced epithelial mesenchymal transition (EMT) in NSCLC cells. CM also enhanced the expression of stem cell markers such as CD44, and increased CD133 mRNA levels in cancer cells and resistance to cisplatin in lung cancer cells as well as CTOS produced cells, indicating that the CM from fibroblasts was biologically active. Furthermore, the concentration of IL-6 was higher in CM from CAFs as compared to that from LNFs, and CM-induced phenotypic changes of cancer cells were inhibited by addition of the IL-6 receptor neutralizing antibody. Cancer cells incubated with IL-6 and TGF-β1 showed significantly increased EMT changes and acquisition of cancer stemness as compared with those treated with TGF-β1 alone, suggesting that IL-6 enhances TGF-β signaling. Cisplatin treatment also increased TGF-β expression in cancer cells, and TGF-β1 activated LNFs and CAFs to increase IL-6 production. Immunohistochemistry was used to examine EMT status, fibroblast distribution, and IL-6 expression in tumor specimens obtained from patients with NSCLC after undergoing chemotherapy. EMT changes of cancer cells were correlated with a diffuse distribution of SMA-stained fibroblasts and stromal IL-6 expression. Conclusion IL-6 from CAFs enhanced epithelial cell EMT and acquisition of cancer stemness by enhancing TGF-β signaling. IL-6 and TGF-β may play contributing roles in maintenance of the paracrine loop between these two cytokines as part of the communication between CAFs and NSCLC cells, resulting in chemoresistance. Citation Format: Yasushi Shintani, Soichiro Funaki, Toru Kimura, Ayako Fujiwara, Tomohiro Kawamura, Toshiya Bessho, Masayoshi Inoue, Masato Minami, Meinoshin Okumura. Activation of TGF β-IL6 axis mediates chemoresistance in non-small cell lung cancer by increasing epithelial-mesenchymal transition signaling. [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 5074. doi:10.1158/1538-7445.AM2015-5074

Ginsenoside metabolite compound K enhances the efficacy of cisplatin in lung cancer cells.

To evaluate the potential of ginsenoside metabolite compound K (CK) in enhancing the anti-tumor effects of cisplatin against lung cancer cells, including cell proliferation and apoptosis, and the underlying mechanism. Western blotting and p53 reporter assay were used to assess p53 expression and activity. MTT assay and TUNEL staining were employed to investigate the drug effects on cell growth and apoptosis, respectively. Combination index (CI) was calculated to determine synergism. We found that CK could significantly enhance cisplatin-induced p53 expression and activity in two lung cancer cell lines, H460 and A549. Consequently, synergistic inhibition of cell growth was observed when the cells were co-treated with CK and cisplatin compared to single treatment. In addition, the ability of cisplatin in apoptosis induction was similarly synergized by CK. Furthermore, by using p53-null lung cancer cells, we demonstrate that the synergy was p53 dependent. Conventional chemotherapies are often accompanied by development of drug resistance and severe side effects. Novel discoveries of low toxicity compounds to improve the outcome or enhance the efficacy of chemotherapies are of great interest. In the present study, our data provide the first evidence that CK could be potentially used as an agent to synergize the efficacy of cisplatin in lung cancer.

FOXO3a mediates the cytotoxic effects of cisplatin in lung cancer cells.

Cisplatin is one of the major chemotherapeutic agents used against different human cancers. A better understanding of the downstream cellular targets of cisplatin will provide information on its mechanism of action. FOXO3a is a member of the FOXO transcription factor family, which modulates the expression of genes involved in cell cycle arrest, apoptosis, and other cellular processes. In this study, we have investigated the effects of cisplatin in a panel of lung cancer cell lines. The results showed that cisplatin inhibited the proliferation of these lung cancer cell lines by inhibiting the PI3K/AKT pathway, with evidence of decreasing phosphorylation of PI3K and AKT under cisplatin treatment, and constitutively activating AKT1 could reduce cisplatin-induced cell apoptosis. More importantly, cisplatin significantly inhibited FOXO3a phosphorylation (at Thr32, AKT phosphorylation site) and induced FOXO3a nuclear accumulation, which in turn increased the expression of FOXO3a-dependent apoptotic protein Bim. Knockdown of FOXO3a expression using small interfering RNA attenuated cisplatin-induced apoptosis. Furthermore, activation of FOXO3a induced cell apoptosis irrespective of p53 status, whereas p53 may act as FOXO3a downstream molecules involved in cisplatin-induced cell apoptosis. Together, our findings suggested that FOXO3a is a relevant mediator of the cytotoxic effects of cisplatin in lung cancer cells.

Synthesis and in vitro study of cisplatin-loaded Fe3O4 nanoparticles modified with PLGA-PEG6000 copolymers in treatment of lung cancer

In the field of cancer therapy, magnetic nanoparticles modified with biocompatible copolymers are promising vehicles for the delivery of hydrophobic drugs such as Cisplatin. The major aim of this effort was to evaluate whether Cisplatin-Encapsulated magnetic nanoparticles improved the anti-tumour effect of free Cisplatin in lung cancer cells. The PLGA-PEG triblock copolymer was synthesised by ring-opening polymerisation of d,l-lactide and glycolide with polyethylene glycol (PEG6000) as an initiator. The bulk properties of these copolymers were characterised using Fourier transform infrared spectroscopy. Cisplatin-loaded nanoparticles (NPs) were prepared by double emulsion solvent evaporation technique and were characterised for size, drug entrapment efficiency (%), drug content (% w/w), and surface morphology. In vitro release profile of cisplatin-loaded NP formulations was determined. Cytotoxic assays were evaluated in lung carcinoma (A549)-treated cells by the MTT assay technique. In addition, the particles were characterised by X-ray powder diffraction, scanning electron microscopy, Fourier transform infrared spectroscopy, and vibrating sample magnetometry. The anti-proliferative effect of Cisplatin appeared much earlier when the drug was encapsulated in magnetic nanoparticles than when it was free. Cisplatin-Encapsulated magnetic nanoparticles significantly enhanced the decrease in IC50 rate. The in vitro cytotoxicity test showed that the Fe3O4-PLGA-PEG6000 magnetic nanoparticles had no cytotoxicity and were biocompatible. The chemotherapeutic effect of free Cisplatin on lung cancer cells is improved by its encapsulation in modified magnetic nanoparticles. This approach has the prospective to overcome some major limitations of conventional chemotherapy and may be a promising strategy for future applications in lung cancer therapy.

Ibuprofen enhances the anticancer activity of cisplatin in lung cancer cells by inhibiting the heat shock protein 70

Hsp70 is often overexpressed in cancer cells, and the selective cellular survival advantage that it confers may contribute to the process of tumour formation. Thus, the pharmacological manipulation of Hsp70 levels in cancer cells may be an effective means of preventing the progression of tumours. We found that the downregulation of Hsp70 by ibuprofen in vitro enhances the antitumoural activity of cisplatin in lung cancer. Ibuprofen prominently suppressed the expression of Hsp70 in A549 cells derived from lung adenocarcinoma and sensitized them to cisplatin in association with an increase in the mitochondrial apoptotic cascade, whereas ibuprofen alone did not induce cell death. The cisplatin-dependent events occurring up- and downstream of mitochondrial disruption were accelerated by treatment with ibuprofen. The increase in cisplatin-induced apoptosis caused by the depletion of Hsp70 by RNA interference is evidence that the increased apoptosis by ibuprofen is mediated by its effect on Hsp70. Our observations indicate that the suppression of Hsp70 by ibuprofen mediates the sensitivity to cisplatin by enhancing apoptosis at several stages of the mitochondrial cascade. Ibuprofen, therefore, is a potential therapeutic agent that might allow lowering the doses of cisplatin and limiting the many challenge associated with its toxicity and development of drug resistance.

Abstract 2057: Cigarette smoke enhances pulmonary carcinogenesis via downregulation of has-miR-487b in respiratory epithelial cells

Abstract Limited information is available regarding the mechanisms by which miRNA alternations contribute to initiation and progression of lung cancer. In order to examine this issue, array techniques were used to assess miRNA expression profiles in short term normal human bronchial epithelia (NHBE) and small airway epithelial cells (SAEC), as well as A549 and Calu-6 lung cancer cells cultured in normal media (NM) with or without cigarette smoke condensate (CSC). Under relevant exposure conditions, CSC consistently mediated a 3.4 − 5.5 fold decrease in has-miR-487b expression in cultured normal as well as lung cancer cells. Quantitative RT-PCR experiments demonstrated that expression levels of has-miR-487b were significantly downregulated (2.9 − 21 fold) in a panel of resected lung cancers relative to adjacent normal tissues. Software-guided analysis revealed numerous potential targets for has-miR-487b including H2AZ, encoding a histone variant modulating expression of developmentally regulated genes as well as SUZ12, which encodes a component of polycomb repressor complexes implicated in aberrant silencing of tumor suppressor genes such as Dickkopf-1 (Dkk-1) in lung cancer cells. Quantitative RT-PCR experiments revealed that constitutive over-expression of has-miR-487b mediated 3-35 fold reductions in H2AZ and SUZ12 expression in SAEC, A549, and Calu-6 cells. RNA cross-link immunoprecipitation (CLIP) experiments confirmed direct interference of has-miR-487b with H2AZ and SUZ12 transcripts. Overexpression of has-miR-487b enhanced sensitivity to cisplatin in lung cancer cells. Collectively, these data suggest that CSC-mediated downregulation of has-miR-487b promotes pulmonary carcinogenesis, in part, via activation of genes encoding proteins implicated in epigenetic regulation of stem cell gene expression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2057.

The role of GRP78 on the resistance to cisplatin in SPCA-1 cell line

背景与目的糖调节蛋白78（GRP78）在肺癌细胞中表达增高并与其对化疗药物VP-16的耐药性有关，而GRP78的表达与其对化疗药物顺铂的耐药性是否相关，研究较少。本研究旨在通过检测GRP78在人肺腺癌细胞SPCA-1中的表达及顺铂作用后的细胞生存率，研究GRP78在肺腺癌细胞顺铂耐药中的作用。方法依据是否应用诱导剂A23187将细胞分为实验组和对照组，采用RT-PCR、Western blot方法检测各组细胞中GRP78在核酸和蛋白水平的表达，利用MTT法测定细胞在顺铂作用下的生存率，分析GRP78在SPCA-1细胞顺铂耐药中的作用。结果A23187可以明显诱导SPCA-1细胞中GRP78在核酸和蛋白水平的表达，且其表达水平与A23187具有一定的浓度依赖性，MTT检测显示：实验组细胞生存率明显低于对照组，亦呈一定的A23187浓度依赖性。结论在人肺腺癌细胞SPCA-1中，诱导剂A23187可以诱导GRP78核酸和蛋白水平的表达，其表达与肺癌细胞在顺铂作用下的生存率呈负相关，表明GRP78可提高SPCA-1细胞对顺铂的敏感性，与肺癌细胞化疗耐药有一定的相关性。