BACKGROUND: Lung cancer currently ranks first globally in both incidence and mortality. Pemetrexed (PMX) serves as a first-line treatment for lung adenocarcinoma (LUAD), but the patients often develop drug resistance during therapy. Milk exosome (mEXO) have the advantages of low immunogenicity, high tissue affinity, and low cost, and mEXO itself has anti-tumor effects. Hyaluronan (HA) naturally bind to CD44, a receptor which is highly expressed in LUAD tissues. This study aims to construct hyaluronan-modified milk exosome (HA-mEXO) and preliminarily investigate their molecular mechanisms for reversing PMX resistance through cellular experiments. METHODS: Exosomes were extracted from milk using high-speed centrifugation, and HA-mEXO was constructed. PMX-resistant A549 and PC-9 cell lines were treated with mEXO and HA-mEXO, respectively. CCK-8 assays, colony formation assays, Transwell assays, and flow cytometry were performed to evaluate proliferation, colony formation, migration, invasion, and apoptosis phenotypes in the treated resistant cell lines. Finally, transcriptomic sequencing, analysis, and cellular functional recovery experiments were conducted to investigate the mechanism by which HA-mEXO reverses PMX resistance in LUAD cells. RESULTS: The expression of CD44 in A549 and PC-9 LUAD drug-resistant cell lines was significantly higher than that in parental cells, and the uptake rate of HA-mEXO by drug-resistant cell lines was significantly higher than that of mEXO. Compared to the mEXO group, HA-mEXO-treated A549 and PC-9 resistant cells exhibited significantly reduced half maximal inhibitory concentration (IC50) values for PMX, markedly diminished clonogenic, migratory, and invasive capabilities, and a significantly increased proportion of apoptotic cells. Western blot analysis revealed that, compared to parental cells, A549 and PC-9 drug-resistant cells exhibited downregulated ZNF516 expression and upregulated ABCC5 expression. Immunofluorescence analysis revealed that HA-mEXO treatment downregulated ABCC5 expression in A549 and PC-9 drug-resistant cells compared to the PBS group, whereas co-treatment with HA-mEXO and ZNF516 knockdown showed no significant change in ABCC5 expression. CONCLUSIONS HA-mEXO carrying ZNF516 suppress ABCC5 expression, thereby enhancing the sensitivity of A549 and PC-9 LAUD drug-resistant cells to PMX.
Humans ; Hyaluronic Acid/chemistry* ; Drug Resistance, Neoplasm/drug effects* ; Exosomes/chemistry* ; Adenocarcinoma of Lung/genetics* ; Pemetrexed/pharmacology* ; Animals ; Lung Neoplasms/pathology* ; Milk/chemistry* ; Cell Proliferation/drug effects* ; Apoptosis/drug effects* ; Cell Line, Tumor ; Hyaluronan Receptors/metabolism*

OBJECTIVETo investigate the sequence-dependent effect of combined use of gemcitabine and pemetrexed on the proliferation of human pancreatic carcinoma cell lines BXPC-3 and PANC-1 in vitro and explore the cellular mechanism.

METHODSMTT assay was used to determine the proliferation of the two cells after addition of the two drugs in different sequences, and the cell cycle changes were analyzed by flow cytometry.

RESULTSBoth gemcitabine (10(-7)-10 mg/ml) and pemetrexed (10(-7)-10 mg/ml) significantly inhibited the proliferation of BXPC-3 and PANC-1 cells in a dose- and time-dependent manner. The effect of combined administration of gemcitabine and pemetrexed on the cell proliferation varied with the order of the drug delivery, and addition of gemcitabine 24 h after pemetrexed administration produced a significant enhancement of the inhibitory effect as compared with simultaneous drug administration (P<0.05) or the administration of the two drugs in a reverse order (P<0.05). Compared with the control group, combined administration of gemcitabine and pemetrexed caused obvious cell cycle arrest at G1 and S phases (P<0.05). Simultaneous administration of the two drugs resulted in significantly reduced G2-phase cells (P<0.05); addition of gemcitabine prior to pemetrexed caused cell cycle arrest in G1 phase (P<0.05), while the reverse caused cell cycle in S phase (P<0.05).

CONCLUSIONBoth gemcitabine and pemetrexed can inhibit the proliferation of BXPC-3 and PANC-1 cells, and their synergetic effect depends on the sequence of their administration. The sequential administration of pemetrexed followed by gemcitabine produces significant synergetic effects against the cell proliferation, which might not be associated with their influence of the cell cycle.

Antimetabolites, Antineoplastic ; pharmacology ; Cell Cycle ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Deoxycytidine ; analogs & derivatives ; pharmacology ; Dose-Response Relationship, Drug ; Drug Synergism ; Glutamates ; pharmacology ; Guanine ; analogs & derivatives ; pharmacology ; Humans ; Pancreatic Neoplasms ; pathology ; Pemetrexed

OBJECTIVETo explore the sensitivity of tumor cell lines with acquired resistance to gefitinib to several chemotherapeutic drugs and provide preclinical basis of available chemotherapy regimens after failure of molecular targeted therapy.

METHODSHuman lung adenocarcinoma cell lines PC9 and PC9/G with acquired resistance to gefitinib were cultured in vitro. The sensitivity to chemotherapeutic drugs and inhibition rate of cell proliferation was determined by MTT assay. Effects of drugs on apoptosis and expression of P-170 were determined by flow cytometry. Difference of gene expression profile between PC9 and PC9/G cells was analyzed by DNA microarray. Western blot was used to test the expression of Akt, phospho-Akt and integrin beta1.

RESULTSThe resistance index of PC9/G cells to cisplatin was about 5.4-fold compared with that of PC9 cells. LY294002 may significantly elevate the sensitivity of PC9/G cells to cisplatin (P < 0.05). PC9/G cells were more sensitive to docetaxel than PC9 cells. No significant difference of sensitivity to pemetrexed was found between these two cell lines. Expression level of P-170 in PC9/G cells was lower than that in PC9 cells. In PC9/G cells, the expression of integrin beta1 and DNA healing gene was high and expression of gene during mitosis was low. The level of expression of Akt, phospho-Akt and integrin beta1 in PC9/G cells was higher than that in PC9 cells.

CONCLUSIONIn PC9/G cells, a cell line with acquired resistance to gefitinib, over-expression of PI3K, integrin and DNA restoration gene and continuous activation of PI3K is found to be correlated with resistance to cisplatin. Docetaxel or pemetrexed is a more reasonable choice than cisplatin for treatment of NSCLC patients who failed to respond to EGFR-TKI.

ATP-Binding Cassette, Sub-Family B, Member 1 ; metabolism ; Antineoplastic Agents ; pharmacology ; Apoptosis ; drug effects ; Carcinoma, Non-Small-Cell Lung ; metabolism ; pathology ; Cell Line, Tumor ; Chromones ; pharmacology ; Drug Resistance, Neoplasm ; Glutamates ; pharmacology ; Guanine ; analogs & derivatives ; pharmacology ; Humans ; Integrin beta1 ; metabolism ; Lung Neoplasms ; metabolism ; pathology ; Morpholines ; pharmacology ; Pemetrexed ; Phosphatidylinositol 3-Kinases ; antagonists & inhibitors ; metabolism ; Proto-Oncogene Proteins c-akt ; metabolism ; Quinazolines ; pharmacology ; Taxoids ; pharmacology