Cisplatin Resistance In Tumor Cells Research Articles

TCF12 induces ferroptosis by suppressing OTUB1-mediated SLC7A11 deubiquitination to promote cisplatin sensitivity in oral squamous cell carcinoma.

Chemotherapy resistance is a major obstacle to effective cancer treatment, and promotion of ferroptosis can suppress cisplatin resistance in tumor cells. TCF12 plays a suppressive role in oral squamous cell carcinoma (OSCC), but whether it participates in the regulation of cisplatin resistance by modulating ferroptosis remains unclear. Here, we found that TCF12 expression was decreased in OSCC cells compared with normal oral cells, and it was reduced in cisplatin (DDP)-resistant OSCC cells compared with parental cells. Moreover, overexpression of TCF12 sensitized DDP-resistant cells to DDP by promoting ferroptosis. Intriguingly, silencing TCF12 reversed the promotion effect of the ferroptosis activator RSL3 on ferroptosis and DDP sensitivity, and overexpressing TCF12 antagonized the effect of the ferroptosis inhibitor liproxstatin-1 on ferroptosis and DDP resistance. Mechanically, TCF12 promoted ubiquitination of SLC7A11 and decreased SLC7A11 protein stability through transcriptional repression of OTUB1, thereby facilitating ferroptosis. Consistently, SLC7A11 overexpression neutralized the promotion effect of TCF12 on ferroptosis and DDP sensitivity. Additionally, upregulation of TCF12 hindered the growth of mouse OSCC xenografts and enhanced the DDP sensitivity of xenografts by inducing ferroptosis. In conclusion, TCF12 enhanced DDP sensitivity in OSCC cells by promoting ferroptosis, which was achieved through modulating SLC7A11 expression via transcriptional regulation of OTUB1.

Endoplasmic reticulum stress and quality control in relation to cisplatin resistance in tumor cells.

The endoplasmic reticulum (ER) is a crucial organelle that orchestrates key cellular functions like protein folding and lipid biosynthesis. However, it is highly sensitive to disturbances that lead to ER stress. In response, the unfolded protein response (UPR) activates to restore ER homeostasis, primarily through three sensors: IRE1, ATF6, and PERK. ERAD and autophagy are crucial in mitigating ER stress, yet their dysregulation can lead to the accumulation of misfolded proteins. Cisplatin, a commonly used chemotherapy drug, induces ER stress in tumor cells, activating complex signaling pathways. Resistance to cisplatin stems from reduced drug accumulation, activation of DNA repair, and anti-apoptotic mechanisms. Notably, cisplatin-induced ER stress can dualistically affect tumor cells, promoting either survival or apoptosis, depending on the context. ERAD is crucial for degrading misfolded proteins, whereas autophagy can protect cells from apoptosis or enhance ER stress-induced apoptosis. The complex interaction between ER stress, cisplatin resistance, ERAD, and autophagy opens new avenues for cancer treatment. Understanding these processes could lead to innovative strategies that overcome chemoresistance, potentially improving outcomes of cisplatin-based cancer treatments. This comprehensive review provides a multifaceted perspective on the complex mechanisms of ER stress, cisplatin resistance, and their implications in cancer therapy.

Ribosome Biogenesis Regulator 1 Homolog (RRS1) Promotes Cisplatin Resistance by Regulating AEG-1 Abundance in Breast Cancer Cells

Many ribosomal proteins are highly expressed in tumors and are closely related to their diagnosis, prognosis and pathological characteristics. However, few studies are available on the correlation between ribosomal proteins and chemoresistance. RRS1 (human regulator of ribosome synthesis 1), a critical nuclear protein involved in ribosome biogenesis, also plays a key role in the genesis and development of breast cancer by protecting cancer cells from apoptosis. Given that apoptosis resistance is one of the causes of the cisplatin resistance of tumor cells, our aim was to determine the relationship between RRS1 and cisplatin resistance in breast cancer cells. Here, we report that RRS1 is associated with cisplatin resistance in breast cancer cells. RRS1 silencing increased the sensitivity of MCF-7/DDP cells to cisplatin and inhibited cancer cell proliferation by blocking cell cycle distribution and enhancing apoptosis. AEG-1 (astrocyte elevated gene-1) promotes drug resistance by interfering with the ubiquitination and proteasomal degradation of MDR1 (multidrug resistance gene 1), thereby enhancing drug efflux. We found that RRS1 binds to and stabilizes AEG-1 by inhibiting ubiquitination and subsequent proteasomal degradation, which then promotes drug efflux by upregulating MDR1. Furthermore, RRS1 also induces apoptosis resistance in breast cancer cells through the ERK/Bcl-2/BAX signaling pathway. Our study is the first to show that RRS1 sensitizes breast cancer cells to cisplatin by binding to AEG-1, and it provides a theoretical basis to improve the efficacy of cisplatin-based chemotherapy.

The role of emodin on cisplatin resistance reversal of lung adenocarcinoma A549/DDP cell.

Exploring drugs that reverse drug resistance and increase the sensitivity of chemotherapy drugs could significantly improve treatment effect of cancer. Our study explored the reversal effect and possible molecular mechanisms of emodin on cisplatin resistance in A549/DDP cells. The IC50 and resistance index of cells were determined by Cell Counting Kit-8 assay. The ability of cell proliferation was evaluated by wound healing assay. Transwell assay was used to detect cell invasion and migration. Apoptosis induction rate was determined by flow cytometry assay and 4',6- diamidino- 2-phenylindole staining. Intracellular concentration was determined by HPLC. Western blot analysis was applied to determine expressions of nuclear factor kappa beta (NF-κB) and its downstream proteins. In this study, we found that the growth inhibitory effect of cisplatin was significantly enhanced by emodin in A549/DDP cells. The combined use of emodin with DDP can effectively promote lung cancer cells apoptosis and inhibit cell migration and invasion. Further investigation indicated that reinforcement effect of emodin and DDP may be associated with inhibition of NF-κB pathway and drug efflux-related proteins such as P-glycoprotein (P-gp), multidrug resistance-associated protein (MRP) and Glutathione S-transferase (GST). The key role of NF-κB was further confirmed by the application of NF-κB inhibitor Ammonium pyrrolidinedithiocarbamate. The intervention of both can significantly increase A549/DDP cell apoptosis and inhibit DDP-induced upregulation of P-gp, MRP and GST. Emodin reverses the cisplatin resistance of tumor cells by down-regulating expression of P-gp, MRP and GST, increasing the intracellular accumulation in A549/DDP cells, and the effect may be associated with the NF-κB pathways.

MAFG-AS1/MAFG positive feedback loop contributes to cisplatin resistance in bladder urothelial carcinoma through antagonistic ferroptosis

Though promoting ferroptosis can reduce cisplatin resistance in tumor cells, ferroptosis and cisplatin resistance in bladder urothelial carcinoma (BUC) following long non-coding RNAs (lncRNAs) is largely unknown. Here, we found the highly expressed lncRNA MAF transcription factor G antisense RNA 1 (MAFG-AS1) in BUC, and its inhibition increased the sensitivity of BUC cells to cisplatin by promoting ferroptosis. Mechanically, binding to iron chaperone poly(rC)-binding protein 2 (PCBP2) facilitated the recruitments of MAFG-AS1 to deubiquitinase ubiquitin carboxyl-terminal hydrolase isozyme L5 (UCHL5), thus stabilizing PCBP2 protein itself. Then PCBP2 was confirmed to interact with ferroportin 1 (FPN1), an iron export protein, leading to inhibition of ferroptosis. Moreover, the expression of MAFG-AS1 was regulated by the transcriptional factor MAFG. Interestingly, MAFG-AS1 stimulated MAFG transcription by recruiting histone acetyltransferase p300 (EP300) to promote the histone 3 at lysine 27 (H3K27ac) at genomic locus of MAFG, forming a MAFG-AS1/MAFG positive feedback loop. In patient samples, higher expression of MAFG-AS1 and MAFG in BUC tissues was significantly correlated with T status and N status, such that MAFG-AS1, MAFG, and the combination of the two were independent prognostic indicators and chemotherapy sensitivity predictive biomarkers for BUC patients. These findings suggest that inhibition of MAFG-AS1 and MAFG can increase the sensitivity of BUC cells to cisplatin through promoting ferroptosis, indicating the novel chemotherapy sensitivity biomarkers and therapeutic target for BUC.

Precisely Assembled Nanoparticles against Cisplatin Resistance via Cancer-Specific Targeting of Mitochondria and Imaging-Guided Chemo-Photothermal Therapy.

Cisplatin resistance in tumor cells is known mainly due to the reduced accumulation of platinum ions by efflux, detoxification by intracellular GSH, and nucleotide excision repair machinery-mediated nuclear DNA repair. In this work, theranostic Pt(IV)-NPs, which are precisely self-assembled by biotin-labeled Pt(IV) prodrug derivative and cyclodextrin-functionalized IR780 in a 1:1 molecular ratio, have been developed for addressing all these hurdles via mitochondria-targeted chemotherapy solely or chemophotothermal therapy. In these nanoparticles, IR780 as a small-molecule dye acts as a mitochondria-targeting ligand to make Pt(IV)-NPs relocate finally in the mitochondria and release cisplatin. As demonstrated by in vitro and in vivo experiments, Pt(IV)-NPs can markedly facilitate cancer-specific mitochondrial targeting, inducing mitochondrial dysfunction and mitochondrial DNA (mtDNA) damage, thus greatly increasing the Pt accumulation, reducing the GSH levels, and avoiding DNA repair machinery in cisplatin-resistant cancer cells (A549R), finally resulting in significant inhibition of A549R tumor growth on animal models by chemotherapy solely. Upon near-infrared irradiation, mitochondria-targeted chemophotothermal synergistic therapy can be realized, further overcoming cisplatin resistance and even eliminating A549R tumors completely. Moreover, such novel Pt(IV)-NPs integrate multimodal targeting (cancer and mitochondria targeting), imaging (near-infrared imaging and photoacoustic imaging), and therapeutic (chemo- and photothermal therapy) moieties in a constant ratio (1:1:1) into a single, reproducible, and structurally homogeneous entity, avoiding nonuniform drug loading and premature leakage as well as the discrete steps of imaging and therapy, which thus is more beneficial for precise therapeutics and future clinical translation.

LncRNA HOTTIP Participates in Cisplatin Resistance of Tumor Cells by Regulating miR-137 Expression in Pancreatic Cancer.

AimThis study aimed to investigate the effect of HOTTIP and miR-137 on cisplatin resistance of pancreatic cancer cells, and study the mechanism of the effect of HOTTIP on the resistance to cisplatin in pancreatic cancer cells, so as to provide new targets for clinical treatment of pancreatic cancer.MethodsPancreatic cancer cells were induced to be resistant to cisplatin by gradually increasing cisplatin concentration at a low concentration gradient in vitro. The changes of HOTTIP and miR-137 were detected, and the effects of HOTTIP and miR-137 on cisplatin efficacy of pancreatic cancer cisplatin-resistant cells were analyzed to explore the mechanism of HOTTIP on cisplatin resistance of pancreatic cancer cells.ResultsAfter inducing cisplatin resistance in pancreatic cancer cells, the expression level of HOTTIP in pancreatic cancer cells further increased and miR-137 decreased. Silencing HOTTIP or over-expression of miR-137 can increase the sensitivity of pancreatic cancer cisplatin-resistant cells to cisplatin, inhibit the proliferation of pancreatic cancer cells, and promote apoptosis. And we found HOTTIP can target to inhibit miR-137 expression. Rescue experiments showed that regulating miR-137 cannot affect the expression of HOTTIP, miR-137 is a downstream target of HOTTIP, and down-regulation of miR-137 expression can obviously hinder the cisplatin sensitization effect of silencing HOTTIP on cisplatin-resistant pancreatic cancer cells.ConclusionSilencing HOTTIP reverses cisplatin resistance of pancreatic cancer cells by promoting miR-137 expression.

Glutathione-Scavenging Poly(disulfide amide) Nanoparticles for the Effective Delivery of Pt(IV) Prodrugs and Reversal of Cisplatin Resistance.

Despite the broad antitumor spectrum of cisplatin, its therapeutic efficacy in cancer treatment is compromised by the development of drug resistance in tumor cells and systemic side effects. A close correlation has been drawn between cisplatin resistance in tumor cells and increased levels of intracellular thiol-containing species, especially glutathione (GSH). The construction of a unique nanoparticle (NP) platform composed of poly(disulfide amide) polymers with a high disulfide density for the effective delivery of Pt(IV) prodrugs capable of reversing cisplatin resistance through the disulfide-group-based GSH-scavenging process, as described herein, is a promising route by which to overcome limitations associated with tumor resistance. Following systematic screening, the optimized NPs (referred to as CP5 NPs) showed a small particle size (76.2 nm), high loading of Pt(IV) prodrugs (15.50% Pt), a sharp response to GSH, the rapid release of platinum (Pt) ions, and notable apoptosis of cisplatin-resistant A2780cis cells. CP5 NPs also exhibited long blood circulation and high tumor accumulation after intravenous injection. Moreover, in vivo efficacy and safety results showed that CP5 NPs effectively inhibited the growth of cisplatin-resistant xenograft tumors with an inhibition rate of 83.32% while alleviating serious side effects associated with cisplatin. The GSH-scavenging nanoplatform is therefore a promising route by which to enhance the therapeutic index of Pt drugs used currently in cancer treatment.

Tetrathiomolybdate inhibits the reaction of cisplatin with human copper chaperone Atox1.

Cisplatin is a widely used anticancer drug in clinic, and ammonium tetrathiomolybdate ([(NH4)2MoS4], TM) is a copper chelator used in clinic for the treatment of Wilson's disease. Recently, TM has been found to enhance the therapeutic effect of cisplatin; however, the origin of this effect is not clear. Here we found that TM can inhibit the reaction of cisplatin with Cu-Atox1 and prevent the protein unfolding and aggregation induced by cisplatin. Although Ag(i) binds to Atox1 in a way similar to Cu(i)-Atox1, TM does not prevent the reaction of Ag-Atox1 with cisplatin. This result indicates that the formation of a Mo-centered trimeric protein cluster in the TM-Cu-Atox1 system plays a role in the inhibitory effect. This work provides new insights into the mechanism by which TM enhances the cytotoxic efficacy of cisplatin and helps to circumvent cisplatin resistance of tumor cells.

ISG15 silencing increases cisplatin resistance via activating p53-mediated cell DNA repair.

Tumor cells frequently evolved resistance to cisplatin that greatly compromises the efficacy of chemotherapy. Identification of the mechanisms underlying drug resistance is important for developing new therapeutic approaches. ISG15 is found to be elevated in many human carcinomas and cancer cell lines. Here, we identified that the expressions of ISG15 and ISG15-conjugating system were downregulated in drug resistant A549/DDP cells compared to drug sensitive A549 cells. Silencing of ISG15 robustly elevated the resistance to cisplatin, suggesting ISG15 plays an important role in cisplatin resistance. Quantitative proteomics identified 1296 differentially expressed proteins between the control and ISG15 knockdown cells, showing that ISG15 silencing upregulated proteins in p53 pathway, adherens junction and nucleotide excision repair (NER) pathway. We also found that ISG15 silencing induced cell cycle arrest through stabilizing p53 and increasing HnRNP K expression, which allowed the prolonged time for cells to repair cisplatin-damaged DNA. Taken together, we proved that ISG15 downregulation activated the DNA damage/repair pathway to enhance cisplatin resistance in tumor cells.

Abstract TMEM-017: COLLAGEN TYPE XI ALPHA 1 (COL11A1) IS A NOVEL STROMAL MEDIATOR OF CHEMORESISTANCE

Abstract Despite an initial satisfactory response to platinum and taxane-based regimens, approximately 80% of ovarian cancer patients will succumb to chemoresistant recurrent disease. Thus, there is a dire need to understand the mechanism by which tumor cells develop resistance to subsequent therapies after first-line cisplatin treatment. We previously identified that collagen type XI, alpha 1 (COL11A1) is a novel biomarker associated with poor survival, metastasis, and chemoresistance in ovarian cancer. COL11A1 is expressed in a subset of α-SMA-positive cancer-associated fibroblasts (CAFs) adjacent to tumor cells. COL11A1 expression in peritumoral CAFs increases during ovarian cancer progression, with the highest expression in recurrent tumors. However, the functional significance of stromal COL11A1 in ovarian cancer progression and chemoresistance is largely unknown. To determine the role of stromal COL11A1 in chemoresistance, we generated COL11A1-overexpressing TRS3 ovarian fibroblasts, co-cultured them with A2780 and OVCAR3 ovarian cancer cells, and measured cancer cell sensitivity to cisplatin. Our data showed that ovarian cancer cells become resistant to cisplatin when cultured in direct contact with COL11A1-overexpressing fibroblasts as well as in fibroblast conditioned media. Furthermore, full-length COL11A1 protein, extracted from A204 cell line, increased cisplatin resistance in ovarian cancer cells, suggesting that COL11A1 is sufficient to induce chemoresistance. Interestingly, culture of cancer cells on full-length COL11A1 protein resulted in increased number of ALDHhigh cancer stem-like cells and activation of Akt, the principal mediator of pro-survival signaling. To further dissect the mechanism by which COL11A1 leads to cisplatin resistance in tumor cells, we performed RNA-seq analysis of tumor cells after COL11A1 overexpression. Our data showed that COL11A1 did not change the levels of genes involved in cisplatin transport and DNA repair pathway in cancer cells, which are well-established mechanisms to confer cisplatin resistance. Rather, COL11A1 increased the expression of pro-inflammatory genes, some of which are known to confer chemoresistance in ovarian cancer. Consistently, our Western data showed that COL11A1 activates NF-kB signaling in cancer cells, a central pathway regulating pro-inflammatory genes. Collectively, our data suggest that COL11A1 contributes to chemoresistance possibly due to an increased survival of ALDHhigh cancer stem-like cells through activation of pro-inflammatory signaling. Citation Format: Miran Rada, Catherine Eldred, Jessica Sage, Sandra Orsulic and Dong-Joo Cheon. COLLAGEN TYPE XI ALPHA 1 (COL11A1) IS A NOVEL STROMAL MEDIATOR OF CHEMORESISTANCE [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr TMEM-017.

F-Box Protein FBXO22 Mediates Polyubiquitination and Degradation of CD147 to Reverse Cisplatin Resistance of Tumor Cells.

Drug resistance remains a major clinical obstacle to successful treatment of cancer. As posttranslational modification is becoming widely recognized to affect the function of oncoproteins, targeting specific posttranslational protein modification provides an attractive strategy for anticancer drug development. CD147 is a transmembrane glycoprotein contributing to chemo-resistance of cancer cells in a variety of human malignancies. Ubiquitination is an important posttranslational modification mediating protein degradation. Degradation of oncoproteins, CD147 included, emerges as an attractive alternative for tumor inhibition. However, the ubiquitination of CD147 remains elusive. Here in this study, we found that deletion of the CD147 intracellular domain (CD147-ICD) prolonged the half-life of CD147 in HEK293T cells, and we identified that CD147-ICD interacts with FBXO22 using mass spectrometry and Western blot. Then, we demonstrated that FBXO22 mediates the polyubiquitination and degradation of CD147 by recognizing CD147-ICD. While knocking down of FBXO22 prolonged the half-life of CD147 in HEK293T cells, we found that FBXO22 regulates CD147 protein turnover in SMMC-7721, Huh-7 and A549 cells. Moreover, we found that the low level of FBXO22 contributes to the accumulation of CD147 and thereafter the cisplatin resistance of A549/DDP cells. To conclude, our study demonstrated that FBXO22 mediated the polyubiquitination and degradation of CD147 by interacting with CD147-ICD, and CD147 polyubiquitination by FBXO22 reversed cisplatin resistance of tumor cells.

Contribution of intracellular ATP to cisplatin resistance of tumor cells.

Decreased cellular accumulation of cisplatin is a frequently observed mechanism of resistance to the drug. Beside passive diffusion, several cellular proteins using ATP hydrolysis as an energy source are assumed to be involved in cisplatin transport in and out of the cell. This investigation aimed at clarifying the contribution of intracellular ATP as an indicator of energy-dependent transport to cisplatin resistance using the A2780 human ovarian adenocarcinoma cell line and its cisplatin-resistant variant A2780cis. Depletion of intracellular ATP with oligomycin significantly decreased cellular platinum accumulation (measured by flameless atomic absorption spectrometry) in sensitive but not in resistant cells, and did not affect cisplatin efflux in both cell lines. Inhibition of Na(+),K(+)-ATPase with ouabain reduced platinum accumulation in A2780 cells but to a lesser extent compared with oligomycin. Western blot analysis revealed lower expression of Na(+),K(+)-ATPase α(1) subunit in resistant cells compared with sensitive counterparts. The basal intracellular ATP level (determined using a bioluminescence-based assay) was significantly higher in A2780cis cells than in A2780 cells. Our results highlight the importance of ATP-dependent transport, among other processes mediated by Na(+),K(+)-ATPase, for cisplatin influx in sensitive cells. Cellular platinum accumulation in resistant cells is reduced and less dependent on energy sources, which may partly result from Na(+),K(+)-ATPase downregulation. Our data suggest the involvement of other ATP-dependent processes beside those regulated by Na(+),K(+)-ATPase. Higher basal ATP level in cisplatin-resistant cells, which appears to be a consequence of enhanced mitochondrial ATP production, may represent a survival mechanism established during development of resistance.

Enhanced cisplatin cytotoxicity by RNA interfering the excision repair cross-complementing gene 1 in lung cancer cell A549/DDP

背景与目的核苷酸切除修复机制可修复顺铂(DDP)造成的DNA损伤，作为其中的关键酶之一，切除修复交叉互补基因1(excision repair cross-complementing gene 1, ERCC1)的表达可能与DDP耐药有关。本研究旨在探讨小干扰RNA沉默ERCC1基因表达对肺腺癌耐药细胞A549/DDP药物敏感性的影响。方法将体外设计合成针对ERCC1的小分子RNA(siRNA)转染A549/DDP细胞，RT-PCR检测ERCC1 mRNA水平；Western blot检测ERCC1蛋白表达的变化；MTT检测RNA干扰后细胞药物敏感性改变。结果ERCC1 RNAi干扰后ERCC1 mRNA表达指数降低，明显低于对照组，干扰后ERCC1 mRNA表达抑制率为90.4%。转染ERCC1 siRNA降低了ERCC1蛋白的表达水平。MTT显示分别用2 μg/mL、4 μg/mL、8 μg/mL、16 μg/mL、32 μg/mL顺铂处理细胞48 h，转染ERCC1 siRNA细胞组较对照组敏感性明显增高，干扰前A549/DDP细胞IC50为12.49 μg/mL，干扰后A549/DDP细胞IC50下降为9.27 μg/mL。结论ERCC1 siRNA干扰可以降低ERCC1的表达水平，并可提高A549/DDP细胞对顺铂的敏感性。

Abstract 2507: Defective Chk2 expression in cisplatin-resistant ovarian tumor cells impedes wild-type p53 function

Abstract The p53 tumor suppressor in its wild-type state correlates with sensitivity of tumor cells to cisplatin, and mutant p53 with resistance. However, resistance is also a significant impediment in tumors harboring wild-type p53. Identifying the mechanism preventing wild-type p53 from inducing cell death becomes vital in understanding resistance. Our studies in an isogenic sensitive-resistant pair of wild-type p53 ovarian tumor models demonstrate that unlike sensitive A2780 cells, the ∼25-fold resistant 2780CP variant cells do not upregulate p21 at 24 hr in response to cisplatin (1 or 5 µM). Since defects in wild-type p53 and chk2 are reported to be mutually exclusive and Chk2 defects are known to impact p53 function, we investigated chk2 expression in the model pair. By immunoblot analysis, A2780 cells robustly expressed Chk2, whereas levels were undetectable in resistant cells. Real-time PCR demonstrated an absence of Chk2 transcripts in resistant cells, and since chronic exposure to decitabine (1 or 5 µM for 5, 7 or 9 days) failed to upregulate Chk2, this suggested that loss of Chk2 expression in 2780CP cells is likely due to a non-epigenetic mechanism. In order to test whether loss of Chk2 indeed contributes to loss of p53 function and also loss of sensitivity to cisplatin, A2780 cells were exposed to Chk2 siRNA (100 nM for 5 hr), which reduced Chk2 levels by >75%. Exposure of siRNA-treated cells to cisplatin (1.0 µM) followed by immunoblot analysis 24 hr later demonstrated an attenuated p21 expression. Similarly, cytotoxic assessment by the MTT assay provided evidence that Chk2-knockdown in A2780 cells increased resistance to cisplatin by about 2-fold. Since the level of cisplatin resistance in 2780CP cells and other similar models is much greater than those seen in resistant cells harboring mutant- or null-p53, we tested the concept that p53 knockdown by siRNA in Chk2-deficient 2780CP cells may actually sensitize these cells. Support for this was provided by a 2-fold decrease in the IC50 of cisplatin. In contrast, the IC50 of cisplatin increased by about 2-fold in p53-knockdown A2780 cells. If Chk2-deficiency contributes to cisplatin resistance in tumor cells harboring wild-type p53, then specific non-cross-resistant platinum analogs must be activating p53 and inducing cell killing in a Chk2-independent manner. This was confirmed by the demonstration that the platinum(IV) analog DAP increased levels of p21 in Chk2-knockdown A2780 cells without loss of cytotoxic potency, and that knockdown of p53 in either sensitive or resistant cells increased resistance to the analog by about 2- to 3-fold. These results demonstrate the relative significance of Chk2 in the mode of action of cisplatin and the analog DAP. (Supported by NCI RO1 CA127263). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2507.

Relationship between ERCC1 expression and cisplatin intervention in human lung adenocarcinoma cell lines

The excision repair cross-complementing gene 1 (ERCC1), which is important in the repair of cisplatin-DNA adducts, is reported to be related to cisplatin resistance in tumor cells. The aim of this study is to investigate the influence of low-dose cisplatin on expression of ERCC1 gene and to confirm the correlation between ERCC1 and cisplatin resistance in human lung adenocarcinoma cell lines. A549 and A549/DDP cell lines were treated with 10 μmol/L cisplatin for 12, 24, 48 and 72 h, or treated with 5, 10, 20, 40 μmol/L cisplatin for 24 h respectively. Then the expression of ERCC1 mRNA and protein was measured by RT-PCR and immunocytohistology SABC assay respectively. The resistance of A549/DDP cells was measured by MTT assay. After treating with 10 μmol/L cisplatin for 12 h, up-regulation of ERCC1 mRNA and protein was observed in A549 cells, then reached the peak levels in 72 h group. After treating with 5 μmol/L cisplatin for 24 h, up-regulation of ERCC1 mRNA and protein was observed in A549 cells, and when treated with 20 μmol/L cisplatin for 24 h, the ERCC1 mRNA and protein reached the peak levels. Comparing with the parental cells, ERCC1 expression increased obviously in A549/DDP cells, which were established by continuous low-dose cisplatin treatment. Up-regulation of ERCC1 expression can be induced by low-dose cisplatin in human lung adenocarcinoma cell line A549, and ECRR1 may play roles in cisplatin resistance.

Modulation of Cellular Response to Cisplatin by a Novel Inhibitor of DNA Polymerase β

DNA polymerase beta (Pol beta) is an error-prone enzyme whose up-regulation has been shown to be a genetic instability enhancer as well as a contributor to cisplatin resistance in tumor cells. In this work, we describe the isolation of new Pol beta inhibitors after high throughput screening of 8448 semipurified natural extracts. In vitro, the selected molecules affect specifically Pol beta-mediated DNA synthesis compared with replicative extracts from cell nuclei. One of them, masticadienonic acid (MA), is particularly attractive because it perturbs neither the activity of the purified replicative Pol delta nor that of nuclear HeLa cell extracts. With an IC50 value of 8 microM, MA is the most potent of the Pol beta inhibitors found so far. Docking simulation revealed that this molecule could substitute for single-strand DNA in the binding site of Pol beta by binding Lys35, Lys68, and Lys60, which are the main residues involved in the interaction Pol beta/single-strand DNA. Selected inhibitors also affect the Pol beta-mediated translesion synthesis (TLS) across cisplatin adducts; MA was still the most efficient. Therefore, masticadienonic acid sensitized the cisplatin-resistant 2008C13*5.25 human tumor cells. Our data suggest that molecules such as masticadienonic acid could be suitable in conjunction with cisplatin to enhance anticancer treatments.

α6 integrin subunit mediates laminin enhancement of cisplatin-induced apoptosis in testicular tumor germ cells

Our study demonstrates that laminin potentiates cisplatin-induced apoptosis in NCCIT, a testicular tumor germ cell line. When cultured on laminin, NCCIT cells displayed a significantly higher susceptibility to cisplatin-induced apoptosis than on plastic or on other ECM components including fibronectin, Type IV collagen and vitronectin. This high cisplatin sensitivity observed on NCCIT cell cultured on laminin was mediated by the alpha6-integrin signaling. The knockdown of the alpha6-integrin subunit by small interfering RNAs suppressed the higher cisplatin-sensitivity supporting the existence of a crosstalk between laminin-alpha6-integrin signaling and cisplatin-induced apoptosis. Our findings indicate that in cisplatin-treated NCCIT cells, the laminin-alpha6-integrin signaling induces the activation of executioner procaspase-3 and -6 as well as apoptosis-inducing factor (AIF) transcription and expression. The ability of integrin-mediated specific stroma-tumor cell interactions to modulate the chemosensitive phenotype of a tumor cell might provide new insights to overcome cisplatin resistance of tumor cells.

Small interfering RNA-induced suppression of ERCC1 enhances sensitivity of human cancer cells to cisplatin

The level of excision repair cross-complementing 1 (ERCC1) gene expression, which is important in the repair of the cisplatin–DNA adducts, is reported to be related to the level of cisplatin resistance in tumor cells. Therefore, ERCC1 is an attractive target to confer increased cellular sensitivity to cisplatin-based chemotherapy. We designed, synthesized, and utilized small interfering RNAs (siRNAs) that were selective for ERCC1 and investigated their effectiveness in altering the repair capacity of the cells to cisplatin–DNA damage as well as the resistance of the cells to cisplatin. Twenty-four and 48 h after transfecting ERCC1 siRNA1 and siRNA2 targeting the two different regions of the ERCC1 transcript, both the ERCC1 mRNA and protein expression were significantly inhibited, whereas the mock or control siRNA had no effect. The suppression of ERCC1 expression in the HeLa S3 cells led to a decrease in the repair activity of cisplatin-induced DNA damage along with a decrease in the cell viability against platinum-based drugs, such as cisplatin, carboplatin, and oxaliplatin. A similar increased sensitivity to cisplatin and decreased repair activity were also observed for siRNA-mediated ERCC1 silencing in the MCF-7 and HCT116 cells. This study is the first to demonstrate the feasibility of utilizing ERCC1 siRNAs to specifically reduce the ERCC1 expression level in human cancer cells and provides direct evidence for the potential use of ERCC1 siRNA as a chemotherapy-sensitizing agent.

Ubiquitous induction of resistance to platinum drugs in human ovarian, cervical, germ-cell and lung carcinoma tumor cells overexpressing isoforms 1 and 2 of dihydrodiol dehydrogenase.

We have recently demonstrated that overexpression of dihydrodiol dehydrogenase (DDH) in human ovarian carcinoma cells (2008/C13*) is associated with cisplatin and carboplatin resistance. Furthermore, we have also elucidated that transfection of parental human ovarian carcinoma cells with a full-length DDH1 cDNA leads to induction of resistance to the platinum drugs. The development of cisplatin resistance in the transfected cells is associated with an increase in DDH enzyme activity. Previous studies have identified several different mechanisms for development of cisplatin resistance, including altered DNA repair capacity, increased GSH-based detoxification, and increased metallothionein content. However, none of these mechanisms has been found to be universally associated with the development of cisplatin resistance in tumor cells from different tissue sources. The present study was undertaken to assess whether overexpression of DDH1 or DDH2 (in human ovarian, cervical, lung and germ-cell tumor cell lines) could specifically induce resistance to the platinum drugs in these cell lines. We demonstrated a ubiquitous association of increased expression of DDH1 or DDH2 (as judged by increased enzyme activity in transfected clones) with development of resistance to cisplatin and carboplatin. Moreover, we also found a lack of cross-resistance to anticancer drugs that have a different mode of action including paclitaxel, vincristine, doxorubicin hydrochloride, and melphalan. Although at present it is not clear how DDH is involved in platinum drug resistance, the identification of this gene as a causal factor in a series of cell lines derived from different tumors with different intracellular compositions indicates the importance of deciphering this hitherto undefined pathway which can produce resistance to platinum drugs.