Inhibition of SMYD2 Sensitized Cisplatin to Resistant Cells in NSCLC Through Activating p53 Pathway.
ABSTRACT: The protein lysine methyltransferase SMYD2 has recently emerged as a new enzyme modulate gene transcription or signaling pathways, and involved into tumor progression. However, the role of SMYD2 in drug resistant is still not known. Here, we found that inhibition of SMYD2 by specific inhibitor could enhance the cell sensitivity to cisplatin (CDDP), but not paclitaxel, NVB, and VCR in non-small cell lung cancer (NSCLC). Further study showed that SMYD2 and its substrates were overexpressed in NSCLC resistant cells, and the inhibition of SMYD2 or knockdown by specific siRNA could reverse the cell resistance to cisplatin treatment in NSCLC/CDDP cells. In addition, our data indicated that the inhibition or knockdown SMYD2 inhibit tumor sphere formation and reduce cell migration in NSCLC/CDDP cells, but not in NSCLC parental cells. Mechanistically, inhibition of SMYD2 could enhance p53 pathway activity and induce cell apoptosis through regulating its target genes, including p21, GADD45, and Bax. On the contrary, the sensitivity of cells to cisplatin was decreased after knockdown p53 or in p53 deletion NSCLC cells. The synergistically action was further confirmed by in vivo experiments. Taken together, our results demonstrate SMYD2 is involved into cisplatin resistance through regulating p53 pathway, and might become a promising therapeutic target for cisplatin resistance in NSCLC.
Project description:Background:Non-small cell lung cancer (NSCLC) is the leading cause of cancer deaths primarily due to chemoresistance. Somatic mutation of TP53 (36%) and epidermal growth factor receptor (EGFR; > 30%) are major contributors to cisplatin (CDDP) resistance. Substantial evidence suggests the elevated levels of reactive oxygen species (ROS) is a key determinant in cancer. The elevated ROS can affect the cellular responses to chemotherapeutic treatments. Although the role of EGFR in PI3K/Akt signaling cascade in NSCLC is extensively studied, the molecular link between EGFR and p53 and the role of ROS in pathogenesis of NSCLC are limitedly addressed. In this study, we investigated the role of p53 in regulation of ROS production and EGFR signaling, and the chemosensitivity of NSCLC. Methods:In multiple NSCLC cell lines with varied p53 and EGFR status, we compared and examined the protein contents involved in EGFR-Akt-P53 signaling loop (EGFR, P-EGFR, Akt, P-Akt, p53, P-p53) by Western blot. Apoptosis was determined based on nuclear morphological assessment using Hoechst 33258 staining. Cellular ROS levels were measured by dichlorofluorescin diacetate (DCFDA) staining followed by flow cytometry analysis. Results:We have demonstrated for the first time that activation of p53 sensitizes chemoresistant NSCLC cells to CDDP by down-regulating EGFR signaling pathway and promoting intracellular ROS production. Likewise, blocking EGFR/PI3K/AKT signaling with PI3K inhibitor elicited a similar response. Our findings suggest that CDDP-induced apoptosis in chemosensitive NSCLC cells involves p53 activation, leading to suppressed EGFR signaling and ROS production. In contrast, in chemoresistant NSCLC, activated Akt promotes EGFR signaling by the positive feedback loop and suppresses CDDP-induced ROS production and apoptosis. Conclusion:Collectively, our study reveals that the interaction of the p53 and Akt feedback loops determine the fate of NSCLC cells and their CDDP sensitivity.
Project description:BACKGROUND:Clinical treatment of non-small cell lung carcinoma (NSCLC) by cisplatin eventually results in drug resistance, which cancer stem cells and autophagy are believed to be involved in. In the present study, we aimed to explore the effect of autophagy-inhibited cancer stem cells in NSCLC. METHODS:Cancer stem cells were identified by CD133 expression levels detected by immunochemistry, real-time polymerase chain reaction, western blot, and flow cytometry. Stemness was detected by sphere-forming assays of tumor cells. Autophagy was determined by LC3-II expression at mRNA and protein levels. The effect of chloroquine (CQ) on autophagy was detected by real-time polymerase chain reaction, western blot and sphere-forming assay in vitro, and tumor growth in male NOD/SCID mice. RESULTS:Cisplatin (CDDP) treatment enhanced CD133+ cell ratios in clinical NSCLC specimens and NSCLC cell line A549. The CD133+ cells enriched by CDDP exhibited higher autophagy levels. Autophagy inhibition by CQ inhibited CD133+ stemness and promoted CDDP efficiency in A549 cells. In addition, the combination of CDDP and CQ treatment significantly inhibited autophagy levels and cancer stem cell proportions in vitro, and dramatically suppressed tumor growth compared with individual agents. CONCLUSION:Autophagy inhibition of cancer stem cells could promote the efficacy of cisplatin against NSCLC.
Project description:The highly refractory nature of non-small cell lung cancer (NSCLC) to chemotherapeutic drugs is an important factor resulting in its poor prognosis. Recent studies have revealed that tumour necrosis factor alpha-induced protein 8 (TNFAIP8) is involved in various biological and pathological processes of cells, but their underlying mechanisms in processes ranging from cancer development to drug resistance have not been fully elucidated.TNFAIP8 expression in clinical NSCLC samples was examined through immunohistochemistry (IHC). After adjusting for patients' characteristics with propensity score matching, Kaplan-Meier analysis and Cox regression analysis were performed for comparison of patients' survival according to the TNFAIP8 level. Lentiviral transfection with TNFAIP8-specific shRNAs was used to establish stable TNFAIP8 knockdown (TNFAIP8 KD) NCI-H460, A549 and cis-diamminedichloroplatinum II resistant A549 (A549/cDDP) cell lines. Cell proliferation and viability were assessed by CCK-8 assay. Cell cycle was examined by flow cytometry. Multiple pathways regulated by TNFAIP8 KD were revealed by microarray analysis.We found that high TNFAIP8 expression was associated with advanced pT stage, advanced pTNM stage, lymph node metastasis and unfavourable survival in NSCLC patients. TNFAIP8 shRNAs reduced in vitro cancer cell proliferation and in vivo tumor growth. Additionally, TNFAIP8 KD increased the sensitivity of NSCLC cells to cisplatin in vitro and in vivo. Conversely, up-regulation of TNFAIP8 promoted the?proliferation and?drug resistance to cisplatin?of NSCLC cells. TNFAIP8 influences cancer progression pathways involving the MDM2/p53 pathway. Indeed, we observed that TNFAIP8 KD mediated the MDM2 downregulation and the p53 ubiquitination, thereby decreasing the degradation of p53 protein. shRNA p53 reversed TNFAIP8 shRNA-mediated regulation of cell proliferation, cell cycle, cisplatin sensitivity, and expression levels of RAD51, a DNA repair gene.Our work uncovers a hitherto unappreciated role of TNFAIP8 in NSCLC proliferation and cisplatin chemoresistance that is mediated through the MDM2/p53 pathway. These findings might offer potential therapeutic targets for reversing cisplatin resistance in NSCLC patients with high TNFAIP8 expression.
Project description:Non-small cell lung cancers (NSCLC) are commonly treated with a platinum-based chemotherapy such as cisplatin (CDDP) in combination with ionizing radiation (IR). Although clinical trials have demonstrated that the combination of CDDP and IR appear to be synergistic in terms of therapeutic efficacy, the mechanism of synergism remains largely uncharacterized. We investigated the role of the DNA damage response (DDR) in CDDP radiosensitization using two NSCLC cell lines. Using clonogenic survival assays, we determined that the cooperative cytotoxicity of CDDP and IR treatment is sequence dependent, requiring administration of CDDP prior to IR (CDDP-IR). We identified and interrogated the unique time and agent-dependent activation of the DDR in NSCLC cells treated with cisplatin-IR combination therapy. Compared to treatment with CDDP or IR alone, CDDP-IR combination treatment led to persistence of γH2Ax foci, a marker of DNA double-strand breaks (DSB), for up to 24h after treatment. Interestingly, pharmacologic inhibition of DDR sensor kinases revealed the persistence of γ-H2Ax foci in CDDP-IR treated cells is independent of kinase activation. Taken together, our data suggest that delayed repair of DSBs in NSCLC cells treated with CDDP-IR contributes to CDDP radiosensitization and that alterations of the DDR pathways by inhibition of specific DDR kinases can augment CDDP-IR cytotoxicity by a complementary mechanism.
Project description:Drug resistance greatly limits the therapeutic efficacy of treatment of non-small cell lung cancer (NSCLC). One of the important factors is the dysfunction of tumor suppressor p53. Recent studies have suggested that p53 suppresses tumors by regulating number of mitochondrial proteins, including peroxisome proliferator-activated receptor coactivator (PGC1?). Although several studies have confirmed the interaction between p53 and PGC1?, the precise mechanism has not been completely determined in NSCLC. In this study, we investigated the specific signaling between p53 and PGC1? to improve anti-tumor drug effects on NSCLC. We found that low expression of p53 and high expression of PGC1? correlated with shorter survival time of NSCLC patients. In vitro experiments confirmed that NCI-H1299 (p53-null) cells had high levels of PGC1? and were insensitive to cisplatin (CDDP). When PGC1? was knocked down, the sensitivity to cisplatin was increased. Notably, the stability of PGC1? is an important mechanism in its activity regulation. We demonstrated that p53 decreased the stability of PGC1? via the ubiquitin proteasome pathway, which was mediated by protein kinase B (AKT) inhibition and glycogen synthase kinase (GSK-3?) activation. Therefore, p53 may regulate the stability of PGC1? through the AKT/GSK-3? pathway, thus affect the chemosensitivity of NSCLC.
Project description:The antineoplastic agent cis-diammineplatinum(II) dichloride (cisplatin, CDDP) is part of the poorly effective standard treatment of non-small cell lung carcinoma (NSCLC). Here, we report a novel strategy to improve the efficacy of CDDP. In conditions in which CDDP alone or either of two PARP inhibitors, PJ34 hydrochloride hydrate or CEP 8983, used as standalone treatments were inefficient in killing NSCLC cells, the combination of CDDP plus PJ34 or that of CDDP plus CEP 8983 were found to kill a substantial fraction of the cells. This cytotoxic synergy could be recapitulated by combining CDDP and the siRNA-mediated depletion of the principal PARP isoform, PARP1, indicating that it is mediated by on-target effects of PJ34 or CEP 8983. CDDP and PARP inhibitors synergized in inducing DNA damage foci, mitochondrial membrane permeabilization leading to cytochrome c release, and dissipation of the inner transmembrane potential, caspase activation, plasma membrane rupture and loss of clonogenic potential in NSCLC cells. Collectively, our results indicate that CDDP can be advantageously combined with PARP inhibitors to kill several NSCLC cell lines, independently from their p53 status. Combined treatment with CDDP and PARP inhibitors elicits the intrinsic pathway of apoptosis.
Project description:Cisplatin is one of the most common chemotherapeutic drugs for non-small cell lung cancer (NSCLC). However, the response rate is limited because of drug resistance. Histone deacetylase inhibitors (HDACis), which can alter DNA accessibility by regulating chromatin structure and inducing apoptosis, exhibit a synergistic action with cisplatin. However, no biomarkers that can predict the efficacy of the combination of HDACis and cisplatin have been reported. Our study found that panobinostat, an HDAC inhibitor, increased the cisplatin sensitivity of several NSCLC cell lines with low ERCC1 expression but not those with high ERCC1 expression or gain-of-function (GOF) p53 mutation despite of ERCC1 expression level. ERCC1 knockdown increased the cisplatin sensitivity of NSCLC cell lines with high ERCC1 expression without GOF p53 mutations. In addition, in low ERCC1 expression NSCLC cell lines, knockdown of GOF mutant p53 enhanced cisplatin sensitivity. Further double knockdown of ERCC1 and GOF mutant p53 but not ERCC1 knockdown alone increased the cisplatin sensitivity of cells with both high ERCC1 expression and GOF p53 mutations. Therefore, this study demonstrated that ERCC1 expression combined with p53 mutation status may determine the efficacy of cisplatin and HDACi combined therapy and guide the development of future NSCLC therapies.
Project description:Background:Lung cancer is the leading cause of cancer deaths in the world. The major histopathological subtype of lung cancer is non-small cell lung cancer (NSCLC). Platinum-based therapy is the standard of care for patients with advanced stage NSCLC. However, even with treatment, most patients will die of this disease within 5 years and most of these deaths are due to recurrence. One strategy to inhibit recurrence is to use cytostatic compounds following courses of lethal chemotherapy. We have shown in various cancer cell types that mifepristone (MF), an anti-progestin/anti-glucocorticoid, is a powerful cytostatic anti-cancer agent. Thus, in this work we tested the hypothesis that MF should be efficacious in inducing cytostasis and preventing repopulation of NSCLC following cisplatin (CDDP) therapy. Methods:We established an in vitro approach wherein human NSCLC cells with different genetic backgrounds and sensitivities to CDDP (A549 and H23) were exposed to rounds of lethal concentrations of CDDP for 1 h followed or not by MF monotherapy. Every 2 days, cell number, cell viability, and colony-forming ability of viable cells were studied. Results:CDDP killed the majority of cells, yet there were remnant cells escaping CDDP lethality and repopulating the culture, as evidenced by the improved clonogenic survival of viable cells. In contrast, when cells exposed to CDDP where further treated with MF following CDDP removal, their number and clonogenic capacity were reduced drastically. Conclusion:This study reports that there is repopulation of NSCLC cells following a lethal concentration of CDDP monotherapy, that NSCLC cells are sensitive to the growth inhibition properties of MF, and that MF abrogates the repopulation of NSCLC cells following CDDP therapy. Our study supports further evaluating MF as an adjuvant therapy for NSCLC.
Project description:BackgroundLong noncoding RNA taurine upregulated 1 (TUG1) has been reported to play an important role in human cancers. However, little is known about the role of TUG1 in drug resistance and its mechanism in tongue squamous cell carcinoma (TSCC).MethodsTwenty-one cisplatin-sensitive or resistant TSCC patients were enrolled in this study. Cisplatin-resistant cells (SCC25/CDDP and CAL27/CDDP) were used for experiments in vitro. Transfection was performed using Lipofectamine 2000 transfection reagent. The levels of TUG1, microRNA-133b (miR-133b) and cysteine-X-cysteine chemokine receptor 4 (CXCR4) were measured by quantitative real-time polymerase chain reaction or western blot. The cisplatin resistance was investigated by cell viability, transwell invasion and apoptosis assays. The interactions among TUG1, miR-133b and CXCR4 were evaluated by luciferase reporter assay and RNA immunoprecipitation. Murine xenograft model was established using the stably transfected CAL27/CDDP cells.ResultsTUG1 expression was elevated in cisplatin-resistant TSCC tissues and cells compared with that in sensitive group and its knockdown inhibited cisplatin resistance to SCC25/CDDP and CAL27/CDDP cells. miR-133b was targeted via TUG1 and its overexpression suppressed cisplatin resistance. Moreover, CXCR4 was a target of miR-133b. CXCR4 silence repressed cisplatin resistance, which was reversed by miR-133b knockdown. The level of CXCR4 protein was decreased by inhibition of TUG1 and recuperated by miR-133b knockdown. Besides, interference of TUG1 attenuated tumor growth by regulating miR-133b and CXCR4 in vivo.ConclusionDownregulation of TUG1 impeded cisplatin resistance in TSCC-resistant cells by mediating miR-133b and CXCR4, indicating TUG1 as a promising target for TSCC chemotherapy.
Project description:Resistance to cisplatin (CDDP) is a major cause of cancer treatment failure, including human breast cancer. The tumor suppressor protein p53 is a key factor in the induction of cell cycle arrest, DNA repair, and apoptosis in response to cellular stimuli. This protein is phosphorylated in serine 15 and serine 20 during DNA damage repair or in serine 46 to induce apoptosis. Resveratrol (Resv) is a natural compound representing a promising chemosensitizer for cancer treatment that has been shown to sensitize tumor cells through upregulation and phosphorylation of p53 and inhibition of RAD51. We developed a CDDP-resistant MCF-7 cell line variant (MCF-7R) to investigate the effect of Resv in vitro in combination with CDDP over the role of p53 in overcoming CDDP resistance in MCF-7R cells. We have shown that Resv induces sensitivity to CDDP in MCF-7 and MCF-7R cells and that the downregulation of p53 protein expression and inhibition of p53 protein activity enhances resistance to CDDP in both cell lines. On the other hand, we found that Resv induces serine 20 (S20) phosphorylation in chemoresistant cells to activate p53 target genes such as PUMA and BAX, restoring apoptosis. It also changed the ratio between BCL-2 and BAX, where BCL-2 protein expression was decreased and at the same time BAX protein was increased. Interestingly, Resv attenuates CDDP-induced p53 phosphorylation in serine 15 (S15) and serine 46 (S46) probably through dephosphorylation and deactivation of ATM. It also activates different kinases, such as CK1, CHK2, and AMPK to induce phosphorylation of p53 in S20, suggesting a novel mechanism of p53 activation and chemosensitization to CDDP.