The enhanced tumor inhibitory effects of gefitinib and L-ascorbic acid combination therapy in non-small cell lung cancer cells.
ABSTRACT: Despite documentation of successful therapy with epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors in patients with lung cancer, the response rate of patients treated with this therapy remains low. The present study investigated whether L-ascorbic acid serves an adjuvant role in vitro when combined with the EGFR tyrosine kinase inhibitor gefitinib (Iressa®) in lung cancer cell lines. A total of three human lung cancer cell lines were used. The antiproliferative effects and changes in the cell cycle and expression of intracellular signaling molecules, including extracellular signal-regulated kinases (Erk), signal transducer and activator of transcription 3 (Stat3) and protein kinase B (Akt), were measured in cells treated with gefitinib and/or L-ascorbic acid at various concentrations. When combined with gefitinib, L-ascorbic acid exhibited an additive effect on cell proliferation in all gefitinib-sensitive and gefitinib-resistant cell lines. A decrement of ~40% was observed with a low dose 0.5 mM L-ascorbic acid and gefitinib in the relatively gefitinib-resistant A549 cell line (85.6±5.4% with gefitinib alone vs. 52.7±7.3% with combination therapy; P=0.046). The downregulation of intracellular signaling cascades, including EGFR, Akt, Erk and Stat3, was also observed. L-Ascorbic acid serves an adjuvant role when administered in combination with gefitinib; however, the degree of inhibition of cell proliferation differs between lung cancer cell lines.
Project description:Persistently activated IL-6/STAT3 pathway promotes acquired resistance to targeted therapy with epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) in non-small-cell lung cancer (NSCLC) treatment. miR-206 has been verified to be dysregulated and plays as a negative regulator in lung cancer. However, whether miR-206 may overcome IL6-induced gefitinib resistance in EGFR-mutant lung cancer remains elusive. In this study, we investigated the role of miR-206 in IL6-induced gefitinib-resistant EGFR-mutated lung cancer cell lines. We showed that forced miR-206 expression restored gefitinib sensitivity in IL6-induced gefitinib-resistant EGFR-mutant lung cancer cells by inhibiting IL6/JAK1/STAT3 pathway. Specifically, mechanistic investigations revealed that miR-206 blocked IL-6/STAT3 signalling via directly targeting the 3'-UTR of intracellular IL-6 messenger RNA. Moreover, IL-6 induced miR-206 down-regulation by reducing the cropping process of primary miR-206 (pri-miR-206) into the Drosha/DGCR8 complex. Taken together, our findings reveal a direct role of miR-206 in regulating IL-6/STAT3 pathway and contrarily activated IL-6/STAT3 signalling mediates the miR-206 maturation process in gefitinib-resistant EGFR-mutant lung cancer cells.
Project description:Epidermal growth factor receptor - tyrosine kinase inhibitor (EGFR-TKI) is the first choice of treatment for advanced non-small cell lung cancer (NSCLC) patients harbouring activating EGFR mutations. However, single agent usually has limited efficacy due to heterogeneous resistant mechanisms of cancer cells. Thus drug combination therapy would offer more benefits by synergistic interactions and avoidance of resistance emergence. In this study, we selected 8 NSCLC cell lines with different genetic characteristics as research models to investigate the efficacy of 4 agents (gefitinib, cetuximab, afatinib and dasatinib) and their combinations. As a single agent, both afatinib and dasatinib showed more inhibition against cell proliferation than gefitinib and cetuximab. Afatinib combined with dasatinib demonstrated significantly high efficacy against 7 gefitinib-resistant NSCLC cell lines. Moreover, it reversed the resistance to the 4 studied single agents in PTEN mutated NSCLC cells. By studying the activity of EGFR, Src and their downstream signalling pathways including PI3K/PTEN/Akt, Ras/Raf/MEK/ERK, Src/FAK and JAK/Stat, we demonstrated the synergistic interaction between afatinib and dasatinib was not only due to their blockage of different signalling pathways but also the complemental inhibition of the related signalling molecules such as Stat3. We also found that the level of Src, Stat3, and MAPK may be useful biomarkers predicating synergism between afatinib and dasatinib for the treatment of gefitinib-resistant NSCLC cells.
Project description:Gefitinib, erlotinib or afatinib are the current treatment for non-small-cell lung cancer (NSCLC) harboring an activating mutation of the epidermal growth factor receptor (EGFR), but less than 5% of patients achieve a complete response and the median progression-free survival is no longer than 12 months. Early adaptive resistance can occur as soon as two hours after starting treatment by activating signal transducer and activation of transcription 3 (STAT3) signaling. We investigated the activation of STAT3 in a panel of gefitinib-sensitive EGFR mutant cell lines, and gefitinib-resistant PC9 cell lines developed in our laboratory. Afatinib has great activity in gefitinib-sensitive as well as in gefitinib-resistant EGFR mutant NSCLC cell lines. However, afatinib therapy causes phosphorylation of STAT3 tyrosine 705 (pSTAT3Tyr705) and elevation of STAT3 and RANTES mRNA levels. The combination of afatinib with TPCA-1 (a STAT3 inhibitor) ablated pSTAT3Tyr705 and down-regulated STAT3 and RANTES mRNA levels with significant growth inhibitory effect in both gefitinib-sensitive and gefitinib-resistant EGFR mutant NSCLC cell lines. Aldehyde dehydrogenase positive (ALDH+) cells were still observed with the combination at the time that Hairy and Enhancer of Split 1 (HES1) mRNA expression was elevated following therapy. Although the combination of afatinib with STAT3 inhibition cannot eliminate the potential problem of a remnant cancer stem cell population, it represents a substantial advantage and opportunity to further prolong progression free survival and probably could increase the response rate in comparison to the current standard of single therapy.
Project description:<h4>Objectives</h4>Oncogenic alterations of epidermal growth factor receptor (EGFR) signaling are frequently noted in non-small cell lung cancer (NSCLC). In recent decades, EGFR tyrosine kinase inhibitors (TKIs) have been developed, although the therapeutic efficacy of these inhibitor is restricted to EGFR-mutant patients. In this study, we investigated that clathrin-mediated EGFR endocytosis hampers the effects of gefitinib and sustains NSCLC cells with wild-type EGFR.<h4>Materials and methods</h4>NSCLC cell lines (H358, Calu-3, SNU-1327, and H1703) were stimulated with the EGF and treated with gefitinib and endocytosis inhibitors (phenylarsine oxide (PAO) and Filipin III). Growth inhibition and apoptosis were evaluated. Immunofluorescence, immunoprecipitation, and western blot assay were performed to investigate EGFR endocytosis and determine the signaling pathway. Xenograft mouse models were used to verify the combination effect of gefitinib and PAO in vivo.<h4>Results</h4>We confirmed the differences in EGFR endocytosis according to gefitinib response in wild-type EGFR NSCLC cell lines. EGFR in gefitinib-sensitive and -refractory cell lines tended to internalize through distinct routes, caveolin-mediated endocytosis (CVE), and clathrin-mediated endocytosis (CME). Interestingly, while suppressing CME and CVE did not affect cell survival in sensitive cell lines significantly, CME inhibition combined with gefitinib treatment decreased cell survival and induced apoptosis in gefitinib-refractory cell lines. In addition, blocking CME in the refractory cell lines led to downregulate of p-STAT3 and inhibit nuclear localization of STAT3 in vivo, combination treatment with gefitinib and a CME inhibitor resulted in tumor regression accompanying apoptosis in xenograft mouse models.<h4>Conclusion</h4>Clathrin-mediated EGFR endocytosis contribute primary resistance of gefitinib treatment and CME inhibition combined with gefitinib could be an option in treatment of wild-type EGFR NSCLC.
Project description:Although epidermal growth factor receptor (EGFR) is often over-expressed in soft tissue sarcoma (STS), a phase II trial using an EGFR inhibitor gefitinib showed a low response rate. This study identified a new secondary resistance mechanism of gefitinib in STS, and developed new strategies to improve the effectiveness of EGFR inhibition particularly by blocking the STAT3 pathway.We demonstrated that seven STS cell lines of diverse histological origin showed resistance to gefitinib despite blockade of phosphorylated EGFR (pEGFR) and downstream signal transducers (pAKT and pERK) in PI3K/AKT and RAS/ERK pathways. Gefitinib exposure was not associated with decrease in the ratio of pSTAT3/pSTAT1. The relative STAT3 abundance and activation may be responsible for the drug resistance. We therefore hypothesized that the addition of a STAT3 inhibitor could overcome the STAT3 escape pathway.We found that the addition of STAT3 inhibitor S3I-201 to gefitinib achieved synergistic anti-proliferative and pro-apoptotic effects in all three STS cell lines examined. This was confirmed in a fibrosarcoma xenografted mouse model, where the tumours from the combination group (418mm3) were significantly smaller than those from untreated (1032mm3) or single drug (912 and 798mm3) groups.Our findings may have clinical implications for optimising EGFR-targeted therapy in STS.
Project description:Gefitinib, an EGFR tyrosine kinase inhibitor, is used to treat non-small cell lung cancer (NSCLC) patients with activating EGFR mutations. However, the resistance to gefitinib eventually emerges in most of the patients. To understand its mechanism, we generated two acquired gefitinib-resistant NSCLC cell lines. The resistant cells have slower growth rates, but are more resistant to apoptosis in the presence of gefitinib, compared with their sensitive counterparts. In addition, our genome-wide transcriptome analysis reveals unexpected pathways, particularly autophagy, are dysregulated in the gefitinib-resistant cells. Autophagy is significantly enhanced in resistant cells. Importantly, inhibition of autophagy reduces gefitinib resistance. Furthermore, the phosphorylation of ERK, the extracellular signal-regulated kinase, is activated in resistant cells. Inhibition of ERK phosphorylation abrogates gefitinib resistance by suppressing autophagy both in vitro and in vivo. These findings establish a link between ERK and autophagy in gefitinib resistance, and suggest that the ERK signaling may serve as the potentially therapeutic target for treating gefitinib resistance in NSCLC patients.
Project description:Tyrosine kinase inhibitors (TKIs) are mostly used in non-small cell lung cancer (NSCLC) treatment. Unfortunately, treatment with Gefitinib for a period of time will result in drug resistance and cause treatment failure in clinic. Therefore, exploring novel compounds to overcome this resistance is urgently required. Here we investigated the antitumor effect of homoharringtonine (HHT), a natural compound extracted from Cephalotaxus harringtonia, on Gefitinib-resistant NSCLC cell lines in vitro and in vivo. NCI-H1975 cells with EGFR T790M mutation are more sensitive to HHT treatment compared with that of A549 cells with wild type EGFR. HHT inhibited cells growth, cell viability and colony formation, as well as induced cell apoptosis through mitochondria pathway. Furthermore, we explored the mechanism of HHT inhibition on NSCLC cells. Higher level of interleukin-6 (IL-6) existed in lung cancer patients and mutant EGFR and TGF? signal requires the upregulation of IL-6 through the gp130/JAK pathway to overactive STAT3, an oncogenic protein which has been considered as a potential target for cancer therapy. HHT reversiblely inhibited IL-6-induced STAT3 Tyrosine 705 phosphorylation and reduced anti-apoptotic proteins expression. Gefitinib-resistant NSCLC xenograft tests also confirmed the antitumor effect of HHT in vivo. Consequently, HHT has the potential in Gefitinib-resistant NSCLC treatment.
Project description:The tyrosine kinase Src is involved in the progression of many cancers. Moreover, inhibiting Src activity has been shown to obstruct several signaling pathways regulated by the EGFR. Thus, Src is a valuable target molecule in drug development. The purpose of this study was to identify compounds that directly or indirectly modulate Src to suppress lung cancer cell growth and motility and to investigate the molecular mechanisms underlying the effects of these compounds.Human non-small cell lung cancer (NSCLC) cell lines (PC9, PC9/gef, A549, and H1975) with different EGFR statuses were tested by cytotoxicity and proliferation assays after AC-93253 iodide treatment. Src and Src-related protein expression in AC-93253 iodide-treated PC9, PC9/gef, and A549 cells were assessed by western blotting. The effects of AC-93253 iodide on cancer cell colony formation, invasion, and migration were assessed in PC9 and PC9/gef cells. The synergistic effects of gefitinib and AC-93253 iodide were evaluated by combination index (CI)-isobologram analysis in gefitinib-resistant cell lines. The efficacy of AC-93253 iodide in vivo was determined using nude mice treated with either the compound or the vehicle.Among the compounds, AC-93253 iodide exhibited the most potent dose-independent inhibitory effects on the activity of Src as well as on that of the Src-related proteins EGFR, STAT3, and FAK. Furthermore, AC-93253 iodide significantly suppressed cancer cell proliferation, colony formation, invasion, and migration in vitro and tumor growth in vivo. AC-93253 iodide sensitized tumor cells to gefitinib treatment regardless of whether the cells were gefitinib-sensitive (PC9) or resistant (H1975 and PC9/gef), indicating that it may exert synergistic effects when used in combination with established therapeutic agents. Our findings also suggested that the inhibitory effects of AC-93253 iodide on lung cancer progression may be attributable to its ability to modulate multiple proteins, including Src, PI3K, JNK, Paxillin, p130cas, MEK, ERK, and EGFR.Our data suggest that AC-93253 iodide inhibits NSCLC cell growth and motility by regulating multiple Src-related pathways. Our findings may facilitate the development of therapeutic strategies and anti-tumor drugs that may be useful for treating lung cancer in the future.
Project description:Background: The efficacy of an EGFR-targeted treatment strategy for non-small cell lung cancer (NSCLC) is reduced by drug resistance. IL-22 enhances tumor growth and induces chemotherapy resistance in human lung cancer cells. The present study elucidated the IL-22-induced mechanism underlying EGFR-tyrosine kinase inhibitor (TKI) resistance in NSCLC. Methods: The plasma and tissues of patients who received EGFR-TKIs were utilized to determine the association between IL-22 expression and gefitinib efficacy. The IL-22 effect on the EGFR/ERK/AKT pathways in NSCLC HCC827 and PC-9 cells was determined using the CCK-8 assay, western blot, and flow cytometric analysis. A PC-9 xenograft model of IL-22 exposure was established. Gefitinib was administered to mice in combination with IL-22 or vehicle. Results: We showed that IL-22 expression was higher in the EGFR-TKI-resistant group compared to EGFR-TKI-sensitive group. IL-22 expression was associated with EGFR-TKI efficacy in plasma. Additional treatment of IL-22 induced gefitinib resistance and reduced apoptosis in PC-9 and HCC827 cell lines. Furthermore, we showed that the effects of IL-22 attributed to p-ERK, p-EGFR, and p-AKT up-regulation. IL-22 neutralizing antibody completely abrogated the effects of IL-22 on apoptosis and AKT/EGFR/ERK signaling. Finally, we showed that IL-22 enhanced tumor growth and induced gefitinib resistance in the PC-9 xenograft model. Moreover, compared with gefitinib alone, the combination of IL-22 and gefitinib led to an increase in Ki67-positive staining and a reduction in TUNEL staining. Conclusions: Our findings indicate that IL-22 plays a role in tumor progression and EGFR-TKI resistance in NSCLC. Thus, IL-22 might serve as a novel biomarker to overcome resistance of EGFR-TKI.
Project description:The role of IL10 in the tumorigenesis of various cancer types is still controversial. Here, we found that increased IL10 levels are correlated with a poor prognosis in lung cancer patients. Moreover, IL10 levels were significantly increased in the lungs and serum of EGFRL858R- and Kras4bG12D-induced lung cancer mice, indicating that IL10 might facilitate lung cancer tumorigenesis. IL10 knockout in EGFRL858R and Kras4bG12D mice inhibited the development of lung tumors and decreased the levels of infiltrating M2 macrophages and tumor-promoting Treg lymphocytes. We also showed that EGF increases IL10 expression by enhancing IL10 mRNA stability, and IL10 subsequently activates JAK1/STAT3, Src, PI3K/Akt, and Erk signaling pathways. Interestingly, the IL10-induced recruitment of phosphorylated Src was critical for inducing EGFR through the activation of the JAK1/STAT3 pathway, suggesting that Src and JAK1 positively regulate each other to enhance STAT3 activity. Doxycycline-induced EGFRL858R mice treated with gefitinib and anti-IL10 antibodies exhibited poor tumor formation. In conclusion, IL10 and EGFR regulate each other through positive feedback, which leads to lung cancer formation.