A nanoparticle-conjugated cancer drug provides a novel strategy for cancer therapy. In this study, we manipulated nanodiamond (ND), a carbon nanomaterial, to covalently link paclitaxel for cancer drug delivery and therapy. Paclitaxel was bound to the surface of 3-5 nm sized ND through a succession of chemical modifications. The ND-paclitaxel conjugation was measured by atomic force microscope and nuclear magnetic resonance spectroscopy, and confirmed with infrared spectroscopy by the detection of deuterated paclitaxel. Treatment with 0.1-50 microg ml(-1) ND-paclitaxel for 48 h significantly reduced the cell viability in the A549 human lung carcinoma cells. ND-paclitaxel induced both mitotic arrest and apoptosis in A549 cells. However, ND alone or denatured ND-paclitaxel (after treatment with strong alkaline solution, 1 M NaOH) did not induce the damage effects on A549 cells. ND-paclitaxel was taken into lung cancer cells in a concentration-dependent manner using flow cytometer analysis. The ND-paclitaxel particles were located in the microtubules and cytoplasm of A549 cells observed by confocal microscopy. Furthermore, ND-paclitaxel markedly blocked the tumor growth and formation of lung cancer cells in xenograft SCID mice. Together, we provide a functional covalent conjugation of ND-paclitaxel, which can be delivered into lung carcinoma cells and preserves the anticancer activities on the induction of mitotic blockage, apoptosis and anti-tumorigenesis.
Modification on the surface of nanometer‐sized diamond by caboxylation (carboxylated nanodiamond, CND) has been applied for the conjugation of biological molecules such as DNA and protein. In this study, we examined the cytotoxicity and detection of CNDs on the A549 human lung epithelial cells. Treatment with 5‐nm or 100‐nm CNDs (0.1–100 μg/ml) did not markedly reduce the cell viability and altered the protein expression profile in A549 cells. Nevertheless, CNDs were accumulated in A549 cells observed by atomic force microscope and scanning confocal microscope. Both 5‐ and 100‐nm CND particles exhibited the green fluorescence and were ingested into cells. However, the 5‐nm CNDs but not the 100‐nm CNDs were uptake into the nuclei of these cells. Moreover, the fluorescence intensities in A549 cells were found concentration‐dependently to increase after treatment with 5‐nm CNDs (10–100 μg/ml) by using flow cytometer analysis. The fluorescence intensity of 5‐nm CND was higher than 100‐nm CNDs on cells at equal concentration of CND's treatment. As a whole, it is proposed that 5‐nm CND is more useful nanomaterial for further biological applications based on their uptake ability, detectable and little toxic properties on human cells.
Nanoprobes provide advantages for real-time monitoring of tumor markers and tumorigenesis during cancer progression and development. Epidermal growth factor receptor (EGFR) is a key protein that plays crucial roles for tumorigenesis and cancer therapy of lung cancers. Here, we show a carbon-based nanoprobe, nanodiamond (ND), which can be applied for targeting EGFR and monitoring tumorigenesis of human lung cancer cells in vitro and in vivo. The optimal fluorescent intensities of ND particles were observed in the human lung cancer cells and nude mice under in vivo imaging system. The fluorescence signal of ND particles can be real-time detected in the xenografted human lung tumor formation of nude mice. Moreover, the ND-conjugated specific EGFR antibody cetuximab (Cet) can track the location and distribution of EGFR proteins of lung cancer cells in vitro and in vivo. ND-Cet treatment increased cellular uptake ability of nanocomposites in the EGFR-expressed cells but not in the EGFR-negative lung cancer cells. Interestingly, single ND-Cet complex can be directly observed on the protein G bead by immunoprecipitation and confocal microscopy. Besides, the EGFR proteins were transported to lysosomes for degradation. Together, this study demonstrates that ND-conjugated Cet can apply for targeting EGFR and monitoring tumorigenesis during lung cancer progression and therapy.
Nanodiamond (ND) is carbon nanomaterial developing for biological applications. We found that ND particles were taken into cells by macropinocytosis and clathrin‐mediated endocytosis pathways. ND particles were equal separating into two daughter cells of cell division approximately. Finally, the cell retained a single ND cluster in cytoplasm after sub‐cultured for several generations. Interestingly, ND clusters were carried inside of cell but without inducing damages after long‐term cell culture. Together, these findings provide that endocytic ND particles are non‐cytotoxic in cell division, which can be applied for the labeling and tracking of cancer cells.
Background: Lung cancer has become one of the leading causes of cancer incidence and mortality worldwide. Non-small cell lung carcinoma (NSCLC) is the most common type among all lung cancer cases. NSCLC patients contained high levels of activating epidermal growth factor receptor (EGFR) mutations, such as exon 19 deletion, L858R and T790M. Osimertinib, a third-generation of EGFR tyrosine kinase inhibitor (EGFR-TKI), has therapeutic efficacy on the EGFR-T790M mutation of NSCLC patients; however, treatment of osimertinib still can induce drug resistance in lung cancer patients. Therefore, investigation of the drug resistance mechanisms of osimertinib will provide novel strategies for lung cancer therapy. Methods: The H1975OR osimertinib-resistant cell line was established by prolonged exposure with osimertinib derived from the H1975 cells. The cell proliferation ability was evaluated by the cell viability and cell growth assays. The cell migration ability was determined by the Boyden chamber assays. The differential gene expression profile was analyzed by genome-wide RNA sequencing. The protein expression and location were analyzed by western blot and confocal microscopy. objective: However, osimertinib would induce drug resistance in lung cancer patients after treatment for a period. Therefore, investigation of the drug resistance mechanisms of osimertinib will provide novel strategies for lung cancer therapy. Results: In this study, we established the osimertinib-resistant H1975 (T790M/L858R) cancer cells, named the H1975OR cell line. The cell growth ability was decreased in the H1975OR cells by comparison with the H1975 parental cells. Conversely, the cell migration ability was elevated in the H1975OR cells. We found the differential gene expression profile of cell proliferation and migration pathways between the H1975OR and H1975 parental cells. Interestingly, the protein levels of phospho-EGFR, PD-L1, E-cadherin and β-catenin were decreased, but the survivin and N-cadherin proteins were increased in the H1975OR drug-resistant cells. method: In this study, we established the osimertinib-drug resistant cell lines, named as the H1975OR, which was derived from the parental H1975 cells (EGFR-T790M/L858R). Besides, we want to know more about the cellular differences between these two cells, thus, we further to sequence H1975 and H1975OR cells by RNA next-generation sequencing (NGS). Furthermore, we also analyzed these two cell lines by western blotting, cell growth assay, DNA sequencing, confocal microscopy, etc. Conclusion: Osimertinib induces the opposite effect of proliferation and migration in the drug resistance of EGFR-T790M lung cancer cells. We suggest that differential gene and protein expressions in the cell proliferation and migration pathways may mediate the drug resistance of osimertinib in lung cancer cells. Understanding the molecular drug-resistant mechanisms of proliferation and migration pathways of osimertinib may provide novel targets and strategies for the clinical treatment of EGFR-TKIs in lung cancer patients. result: We found that the expression of p-EGFR (Tyr 1068) was reduced in the H1975OR cells by comparison with parental cells. Moreover, the cell growth ability was decreased in the H1975OR cells by comparison with the parental cells. Conversely, the cell migration ability was increased in the H1975OR cells. Besides, we found that the expression of PD-L1 was significantly decreased in H1975OR drug-resistant cells. Interestingly, we found that the PD-L1 protein expression and location not only located at the cell membrane, but also in nuclear by immunofluorescence staining and confocal microscopy. conclusion: In summary, this study suggests that osimertinib drug resistance lung cancer cells may display various drug resistance molecules of cell survival and migration pathways after treatment with osimertinib. Understanding the drug-resistant mechanisms of osimertinib may provide novel targets and strategies for the clinical treatment of EGFR-TKI in lung cancer patients.
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