Shaorong Yu

Exosomes: decreased sensitivity of lung cancer A549 cells to cisplatin.

Exosomes are small extracellular membrane vesicles of endocytic origin released by many cells that could be found in most body fluids. The main functions of exosomes are cellular communication and cellular waste clean-up. This study was conducted to determine the involvement of exosomes in the regulation of sensitivity of the lung cancer cell line A549 to cisplatin (DDP). When DDP was added to A549 cells, exosomes secretion was strengthened. Addition of the secreted exosomes to other A549 cells increased the resistance of these A549 cells to DDP. Upon exposure of A549 to DDP, the expression levels of several miRNAs and mRNAs reportedly associated with DDP sensitivity changed significantly in exosomes; these changes may mediate the resistance of A549 cells to DDP. Exosomes released by A549 cells during DDP exposure decreased the sensitivity of other A549 cells to DDP, which may be mediated by miRNAs and mRNAs exchange by exosomes via cell-to-cell communication. Although the detailed mechanism of resistance remains unclear, we believed that inhibition of exosomes formation and release might present a novel strategy for lung cancer treatment in the future.

Tumor-derived exosomes in cancer progression and treatment failure

Exosomes have diameter within the range of 30-100nm and spherical to cup-shaped nanoparticles with specific surface molecular characteristics, such as CD9 and CD63. These vesicles are present in nearly all human body fluids, including blood plasma/serum, saliva, breast milk, cerebrospinal fluid, urine, semen, and particularly enriched in tumor microenvironment. Exosomes contain multiple proteins, DNA, mRNA, miRNA, long non-coding RNA, and even genetic materials of viruses/prions. These materials are biochemically and functionally distinct and can be transferred to a recipient cell where they regulate protein expression and signaling pathways. Recently, exosomes are demonstrated to have a close relationship with tumor development and metastasis. Exosomes influence therapeutic effect in cancer patients. In this review, we describe the biogenesis, composition, and function of exosomes. The mechanism on how tumor-derived exosomes contribute to cancer progression and clinical treatment failure is also described, with special focus on their potential applications in cancer therapy.

Cisplatin-resistant lung cancer cell–derived exosomes increase cisplatin resistance of recipient cells in exosomal miR-100–5p-dependent manner

Exosomes derived from lung cancer cells confer cisplatin (DDP) resistance to other cancer cells. However, the underlying mechanism is still unknown. A549 resistance to DDP (A549/DDP) was established. Microarray was used to analyze microRNA (miRNA) expression profiles of A549 cells, A549/DDP cells, A549 exosomes, and A549/DDP exosomes. There was a strong correlation of miRNA profiles between exosomes and their maternal cells. A total of 11 miRNAs were significantly upregulated both in A549/DDP cells compared with A549 cells and in exosomes derived from A549/DDP cells in contrast to exosomes from A549 cells. A total of 31 downregulated miRNAs were also observed. miR-100–5p was the most prominent decreased miRNA in DDP-resistant exosomes compared with the corresponding sensitive ones. Downregulated miR-100–5p was proved to be involved in DDP resistance in A549 cells, and mammalian target of rapamycin (mTOR) expression was reverse regulated by miR-100–5p. Exosomes confer recipient cells’ resistance to DDP in an exosomal miR-100–5p-dependent manner with mTOR as its potential target both in vitro and in vivo. Exosomes from DDP-resistant lung cancer cells A549 can alter other lung cancer cells’ sensitivity to DDP in exosomal miR-100–5p-dependent manner. Our study provides new insights into the molecular mechanism of DDP resistance in lung cancer.

Modified PNA-PCR method A convenient and accurate method to screen plasma KRAS mutations of cancer patients

KRAS mutations are proved to confer dramatic resistance to EGFR target therapy of cancer patients. The aim of this study was to establish a convenient and accurate method to screen plasma KRAS mutations of cancer patients since tumor specimens were not always available in clinical practice. A modified PNA-PCR method was established and evaluated in plasma of 19 pancreatic cancer patients. Our results showed that the modified PNA-PCR assay was a sensitive (87.5%, 14/16) and accurate (92.9%, 13/14) method to screen plasma KRAS mutations of pancreatic cancer patients and there was a high consistency of KRAS mutation status between plasma samples and tumor specimens. The modified protocol could not only screen plasma KRAS mutations rapidly and accurately but also had potential to quantify KRAS mutant DNA to predict treatment response of cancer patients and monitor disease progression. It’s should be indicated that this modified assay was only confirmed in the pancreatic cancer patients in this study and need to be verified in other cancer patients.

Comparative analysis of microRNA expression profiles between A549, A549/DDP and their respective exosomes

Exosomes were reported to transport bioactive molecules and influence the biology behavior of recipient cells. In order to study the role of exosomal microRNAs in the mechanism of cisplatin resistance to lung cancer cells, we analyzed the expression profiles of microRNAs in A549, A549/DDP cells and their exosomes by microarray. The results showed that a certain proportion of microRNAs were co-expressed in the cells and exosomes. Linear regression analysis showed that the expression of microRNAs in A549 and A549/DDP cells were strongly correlated with those in their respective exosomes. The expression level of 5 microRNAs (miR-197-5p, miR-4443, miR-642a-3p, miR-27b-3p and miR-100-5p) with the most differential expression were verified by qRT-PCR. The results were consistent with those of the microarray. Target gene prediction and pathway analysis discovered that the microRNAs in the intersections may participate in drug resistance. And the prediction of their association with diseases found that most of these microRNAs was associated with lung cancer. We could draw a preliminary conclusion that microRNAs in exosomes may be involved in the drug resistance of lung cancer cells to cisplatin.

Epithelial-to-mesenchymal transition correlates with gefitinib resistance in NSCLC cells and the liver X receptor ligand GW3965 reverses gefitinib resistance through inhibition of vimentin

The role of epithelial-to-mesenchymal transition in cancer drug resistance is increasingly acknowledged. We examined whether epithelial-to-mesenchymal transition affects gefitinib resistance in non-small cell lung cancer (NSCLC) cells. Cell viability was detected by CCK-8 assay, VIM expression levels were determined by quantitative real-time polymerase chain reaction. Western blot and immunocytochemistry were performed to determine the protein expression level of vimentin. We observed morphologic differences between gefitinib-sensitive and -insensitive cells. Compared with the sensitive parental cell line, HCC827, vimentin expression levels were increased in HCC827 cells with acquired gefitinib resistance. Vimentin expression was also markedly upregulated in cells with intrinsic gefitinib resistance, and upregulated vimentin expression was correlated with gefitinib sensitivity. Our previous study demonstrated that coadministration of gefitinib and GW3965 resulted in decreased cell proliferation and induced apoptosis. Therefore, we investigated the relationship among GW3965, vimentin, and gefitinib resistance in NSCLC cells by analysis of the expression of vimentin in cells treated with a combination of gefitinib and GW3965. Gefitinib treatment led to increased levels of intracellular vimentin, while combined treatment with gefitinib and GW3965 resulted in decreased vimentin expression levels through reduction of gefitinib drug resistance in NSCLC cells. Overall, these findings suggest that vimentin expression is associated with sensitivity to gefitinib, and our study highlights the potential usefulness of the drug, GW3965, for reversal of gefitinib resistance through inhibition of vimentin expression.

Liver X receptors agonist GW3965 re-sensitizes gefitinib-resistant human non-small cell lung cancer cell to gefitinib treatment by inhibiting NF-κB in vitro

The recent research shows that the inhibition of the nuclear factor-κB (NF-κB) pathway is a promising therapeutic option for patients who progress after treatment with the novel mutant-selective EGFR-TKIs. For propose to find a nontoxic drug to reverse the acquired gefitinib resistance, we examined whether the Liver X Receptors agonist GW3965 affect gefitinib resistance of HCC827/GR-8-2 cells. Cell viability was measured by CCK-8 assay. Levels of NF-κB, p-AKT and caspases were detected by Western blot analysis. Immunocytochemical analysis was used to detect the expression of NF-κB, p-AKT intracellularly. Induction of apoptosis and cell cycle arrest was measured by Flow cytometry assay. And results revealed that more than 90% of HCC827/GR-8-2 cells lived upon treatment with gefitinib at a dose of 5μM for 48h. However, when under the combine treatment of GW3965 (5μM) & gefitinib(5μM), cell death rate was increased observably. Co-administration of gefitinib & GW3965 induced cell apoptosis and cell cycle arrest. Additionally, we observed a dose-dependent- down-regulation of NF-κB in HCC827/GR-8-2 cells treated with gefitinib & GW3965. GW3965 and gefitinib synergistically decreased cell proliferation and induced apoptosis by inhibiting NF-κB signaling pathway in gefitinib resistant cells. These findings support our hypothesis that GW3965 could act as a useful drug to reverse the gefitinib resistance.

Comparison of nedaplatin-based versus cisplatin-based chemotherapy for advanced non-small cell lung cancer among East Asian populations: A meta-analysis

Whether nedaplatin and cisplatin are equally effective for advanced non-small cell lung cancer (NSCLC) remains uncertain. Therefore, we performed a meta-analysis of trials to compare nedaplatin-based chemotherapy with cisplatin-based chemotherapy. We conducted a literature search to identify trials that had investigated the substitution of nedaplatin for cisplatin in the treatment of advanced NSCLC. Fourteen randomized controlled trials were included. We found equivalent overall response, overall survival, and survival probability (0.5-year, 1-year). Considering the toxicity profiles, nausea and vomiting were common in the cisplatin group (OR = 0.28, 95% CI = 0.20–0.40, P < 0.001), whereas severe thrombocytopenia was common in the nedaplatin group (OR = 1.68, 95% CI = 1.18–2.40, P = 0.005). A subgroup analysis of grades 1–4 nephrotoxicity showed that cisplatin-based chemotherapy resulted in more renal toxicity (OR = 0.40, 95% CI = 0.24–0.68, P = 0.001). No significant heterogeneity and publication bias were observed. Cumulative analysis found a stable time-dependent trend. Consistent results stratified by age, regimen, and country were observed. Cisplatin-based chemotherapy was associated with non-inferior antitumor efficacy compared with nedaplatin-based therapy. Therefore, the toxicity profile might play an important role in choosing between cisplatin-based or nedaplatin-based regimens.

Genome-wide profiling of micro-RNA expression in gefitinib-resistant human lung adenocarcinoma using microarray for the identification of miR-149-5p modulation

To understand the mechanism involved in gefitinib resistance, we established gefitinib-resistant human HCC827/GR-8-1 cell line from the parental HCC827 cell line. We compared the micro-RNA expression profiles of the HCC827 cells HCC827/GR-8-1 using Agilent micro-RNA microarrays. The micro-RNAs, such as the miR-149-5p, were up- or downregulated and associated with acquired gefitinib resistance. Quantitative real-time polymerase chain reaction was then performed to verify the expression patterns of different micro-RNAs. The result showed that miR-149-5p was upregulated in the HCC827/GR-8-1 cell line. To investigate the biological function of miR-149-5p in non–small cell lung cancer cells acquired gefitinib resistance, we examined cell proliferation using a cell counting kit-8 assay. Cell viability was evaluated after the miR-149-5p mimics, inhibitors, and negative control were separately transfected into the non–small cell lung cancer cells. The results showed that the non–small cell lung cancer cells transfected with miR-149-5p mimics exhibited reduced cell motility. The drug-sensitivity assay results revealed that the overexpression of miR-149-5p effectively evaluates the half maximal inhibitory concentration values of the cell in response to gefitinib, and the downregulation of miR-149-5p can attenuate the half maximal inhibitory concentration values of the cell lines in response to gefitinib. Furthermore, the levels of miR-149-5p in the HCC827 and HCC827/GR-8-1 cells were inversely correlated with caspase-3 expression. In conclusion, this study revealed that miR-149-5p is upregulated in the HCC827/GR-8-1 cells and involved in the acquired gefitinib resistance.

Liver X receptor agonist T0901317 reverses resistance of A549 human lung cancer cells to EGFR-TKI treatment.

Epidermal growth factor receptor‐tyrosine kinase inhibitor (EGFR‐TKI) is effective in lung cancer patients carrying sensitive EGFR mutations. In this study, we investigated if liver X receptor (LXR) agonist T0901317 could reverse the resistance of lung cancer cell lines A549 and H1650 to EGFR‐TKI treatment. We found that T0901317 could make natural EGFR‐TKI‐resistant A549 human lung cancer cells sensitive to EGFR‐TKI treatment and that this was dependent on LXRβ expression. However, T0901317 does not have a similar effect on another natural EGFR‐TKI‐resistant cell line H1650.