Paclitaxel sensitization of multidrug-resistant cells to chemotherapy is independent of the cell cycle

BACKGROUND: Recent studies have shown that paclitaxel (Taxol) is an active chemotherapeutic in the treatment of small cell lung cancer. Paclitaxel binds to tubulin and prevents depolymerization. This causes cells to arrest in the G(2)/M phase of the cell cycle, resulting in sensitization of cells to drug or radiation treatment. METHODS: A drug-resistant H69 small cell lung cancer subline was established. Cytotoxicity of cisplatin and chlorambucil was determined using the MTT cell viability assay and distribution of DNA in the cell cycle. DNA distribution was analyzed by flow cytometry after treatment with paclitaxel or the other tubulin-binding drugs, vinblastine and navelbine. RESULTS: The H69-EPR drug-resistant subline was resistant to epirubicin (sixfold) and was cross-resistant to cisplatin (7.5-fold) and chlorambucil (7.5-fold). Pretreatment with paclitaxel or vinblastine, but not navelbine, sensitized the subline to cisplatin and chlorambucil (P < 0.05), with no effect on parental H69 cells. Sensitization was dose dependent and occurred at doses below those that caused a G(2)/M block in the cell cycle. CONCLUSION: Sensitization of drug-resistant cells by paclitaxel was not associated with its ability to cause a G(2)/M block in the cell cycle. Sensitization by paclitaxel and vinblastine, but not navelbine, which preferentially targets mitotic tubulin, suggests that sensitization may involve changes in the tubulin-dependent intracellular transport processes rather than changes in mitotic tubulin and the G(2)/M block.     

The 1991 Merck Frosst Award. Multidrug resistance in small cell lung cancer.

The two-year survival rate of patients with small cell lung cancer is less than 10%. The major reason for this poor outcome is the development of drug resistance. Panels of small cell lung cancer cell lines have been established, providing models for the study of drug resistance in this tumour. One such model is the doxorubicin-selected H69AR cell line. H69AR displays the typical multidrug resistance phenotype in that it is cross-resistant to anthracyclines, Vinca alkaloids (e.g., vinblastine) and epipodophyllotoxins (e.g., VP-16). However, H69AR cells do not overexpress P-glycoprotein, the membrane drug efflux pump frequently found on multidrug resistant cells. Some alterations in glutathione levels and associated enzyme activities were found but the data do not support the notion that enhanced drug detoxication is involved in H69AR cell resistance. Fewer drug-induced DNA strand breaks, reduced levels of topoisomerase II, and reduced formation of drug-stabilized DNA/topoisomerase II complexes were observed in H69AR cells. These data implicate topoisomerase II in the resistance phenotype of H69AR cells, but cannot explain H69AR cell resistance to the Vinca alkaloids, which do not have topoisomerase II as a target. Monoclonal antibodies against antigens overexpressed on H69AR cells have been derived and four have been characterized. Immunoscreening of an H69AR cDNA expression library has allowed the identification of one of these antigens as p36 (annexin II), a Ca2+/phospholipid binding protein. Chemosensitizers and novel xenobiotics have been examined for their ability to circumvent the drug resistance of H69AR cells. The limited success of these investigations suggests that innovative approaches may be required. In conclusion, the data obtained with H69AR and other models of small cell lung cancer indicate that multiple mechanisms contribute to drug resistance in this disease.     

Resistance to paclitaxel in a cisplatin-resistant ovarian cancer cell line is mediated by P-glycoprotein

The IGROVCDDP cisplatin-resistant ovarian cancer cell line is also resistant to paclitaxel and models the resistance phenotype of relapsed ovarian cancer patients after first-line platinum/taxane chemotherapy. A TaqMan low-density array (TLDA) was used to characterise the expression of 380 genes associated with chemotherapy resistance in IGROVCDDP cells. Paclitaxel resistance in IGROVCDDP is mediated by gene and protein overexpression of P-glycoprotein and the protein is functionally active. Cisplatin resistance was not reversed by elacridar, confirming that cisplatin is not a P-glycoprotein substrate. Cisplatin resistance in IGROVCDDP is multifactorial and is mediated in part by the glutathione pathway and decreased accumulation of drug. Total cellular glutathione was not increased. However, the enzyme activity of GSR and GGT1 were up-regulated. The cellular localisation of copper transporter CTR1 changed from membrane associated in IGROV-1 to cytoplasmic in IGROVCDDP. This may mediate the previously reported accumulation defect. There was decreased expression of the sodium potassium pump (ATP1A), MRP1 and FBP which all have been previously associated with platinum accumulation defects in platinum-resistant cell lines. Cellular localisation of MRP1 was also altered in IGROVCDDP shifting basolaterally, compared to IGROV-1. BRCA1 was also up-regulated at the gene and protein level. The overexpression of P-glycoprotein in a resistant model developed with cisplatin is unusual. This demonstrates that P-glycoprotein can be up-regulated as a generalised stress response rather than as a specific response to a substrate. Mechanisms characterised in IGROVCDDP cells may be applicable to relapsed ovarian cancer patients treated with frontline platinum/taxane chemotherapy.     

Overexpression of IL-6 but not IL-8 increases paclitaxel resistance of U-2OS human osteosarcoma cells

The cytokines IL-6, initially recognized as a regulator of immune and inflammatory response and IL-8, a potential regulator of angiogenesis, also regulate the growth of many tumor cells. Human cancer cells selected for multidrug resistance to common chemotherapeutic agents demonstrate increased expression of IL-6 and IL-8. To determine whether IL-6 or IL-8 overexpression contributes directly to the drug resistant phenotype, IL-6 or IL-8 cDNA were introduced into the paclitaxel sensitive human osteosarcoma cell line U-2OS using the pIRESneo bicistronic expression vector. Interleukin-6 and IL-8 transfectants were selected for either high IL-6 or IL-8 secretion and evaluated in drug resistance assays. Two IL-6 and two IL-8 secreting clones express IL-6 or IL-8 levels of 10 ng/ml and 1 ng/ml in culture, while parental U-2OS and pIRESneo vector transfected control cells express IL-6 and IL-8 levels of 0.005 ng/ml and 0.1 ng/ml, respectively. MTT cytotoxicity with IL-6 transfected cells demonstrates a five-fold increase in resistance to paclitaxel and a four-fold increase in resistance to doxorubicin as compared to U-2OS. There are no changes in mitoxantrone or topotecan resistance in the IL-6 transfectants as compared to parental U-2OS. Northern analysis of IL-6 transfectants demonstrates that the resistant phenotype is not related to increased levels of MDR-1, MRP-1, or LRP. Western analysis also confirms that P-glycoprotein levels are not altered in IL-6 transfectants. Further supporting an MDR-1 independent mechanism of drug resistance, verapamil cannot reverse paclitaxel resistance in transfected cells, findings further supported by rhodamine 123 exclusion data. Treatment of IL-6 transfected cells with paclitaxel, compared with drug-sensitive parental U-2OS, shows U-2OS(IL-6) are significantly more resistant to apoptosis induced by paclitaxel and exhibit decreased proteolytic activation of caspase-3. In contrast U-2OS(IL-8) transfectants demonstrate no appreciable increase in paclitaxel resistance when compared with parental cells. In summary, while both IL-6 and IL-8 are overexpressed in paclitaxel resistant cell lines, only IL-6 has the potential to contribute directly to paclitaxel and doxorubicin resistance in U-2OS. This resistance is through a non-MDR-1 pathway.     

Differential activation of NF-κB signaling is associated with platinum and taxane resistance in MyD88 deficient epithelial ovarian cancer cells

Development of chemoresistance is a major impediment to successful treatment of patients suffering from epithelial ovarian carcinoma (EOC). Among various molecular factors, presence of MyD88, a component of TLR-4/MyD88 mediated NF-κB signaling in EOC tumors is reported to cause intrinsic paclitaxel resistance and poor survival. However, 50–60% of EOC patients do not express MyD88 and one-third of these patients finally relapses and dies due to disease burden. The status and role of NF-κB signaling in this chemoresistant MyD88^negative population has not been investigated so far. Using isogenic cellular matrices of cisplatin, paclitaxel and platinum-taxol resistant MyD88^negative A2780 ovarian cancer cells expressing a NF-κB reporter sensor, we showed that enhanced NF-κB activity was required for cisplatin but not for paclitaxel resistance. Immunofluorescence and gel mobility shift assay demonstrated enhanced nuclear localization of NF-κB and subsequent binding to NF-κB response element in cisplatin resistant cells. The enhanced NF-κB activity was measurable from in vivo tumor xenografts by dual bioluminescence imaging. In contrast, paclitaxel and the platinum-taxol resistant cells showed down regulation in NF-κB activity. Intriguingly, silencing of MyD88 in cisplatin resistant and MyD88^positive TOV21G and SKOV3 cells showed enhanced NF-κB activity after cisplatin but not after paclitaxel or platinum-taxol treatments. Our data thus suggest that NF-κB signaling is important for maintenance of cisplatin resistance but not for taxol or platinum-taxol resistance in absence of an active TLR-4/MyD88 receptor mediated cell survival pathway in epithelial ovarian carcinoma.     

Epigallocatechin-3-gallate delivers hydrogen peroxide to induce death of ovarian cancer cells and enhances their cisplatin susceptibility

The green tea polyphenol epigallocatechin-3-gallate (EGCG) has cancer chemopreventive properties against various types of cancers. The compound is known to attack various targets in transformed cells. In this report, we examined the action of EGCG on ovarian cancer cells. Eight ovarian cancer cell lines were tested (SKOV3, CAOV3, OVCAR3, OVCAR10, A2780, CP70, C30, and C200) and showed IC50s for EGCG at the micromolar range, including ones that are resistant to the chemotherapeutic drug cisplatin. The ovarian cancer cells were sensitive to H2O2 at similar concentrations, and EGCG treatment led to enhanced intracellular H2O2. Neutralization with pyruvate, a scavenger of H2O2, suggests that the toxicity of EGCG may be mediated by oxidative stress from the free radical. Addition of Tempol, a superoxide dismutase mimetic, demonstrates that H2O2 might be generated endogenously from superoxide. The toxicity of cisplatin and the development of cisplatin resistance are major obstacles in treatment of ovarian cancer. We found that addition of EGCG amplified the toxicity of cisplatin. EGCG increased cisplatin potency by three to six-fold in SKOV3, CAOV3, and C200 cells, the latter being a cell line induced to have several hundred fold resistant to cisplatin above the parental line. Our findings suggest that EGCG may accentuate oxidative stress to inhibit growth of ovarian cancer cells and sensitize them to cisplatin.     

RACK1 and CIS mediate the degradation of BimEL in cancer cells

RACK1 is a 7-WD motif-containing protein with numerous downstream effectors regulating various cellular functions. Using a yeast two-hybrid screen, we identified dynein light chain 1 as a novel interacting partner of RACK1. Additionally, we demonstrated that RACK1 formed a complex with DLC1 and Bim, specifically BimEL, in the presence of apoptotic agents. Upon paclitaxel treatment, RACK1, DLC1, and CIS mediated the degradation of BimEL through the ElonginB/C-Cullin2-CIS ubiquitin-protein isopeptide ligase complex. We further showed that RACK1 conferred paclitaxel resistance to breast cancer cells in vitro and in vivo. Finally, we observed an inverse correlation between CIS and BimEL levels in both ovarian and breast cancer cell lines and specimens. Our study suggests a role of RACK1 in protecting cancer cells from apoptosis by regulating the degradation of BimEL, which together with CIS could play an important role of drug resistance in chemotherapy.     

Positive correlation between cellular glutathione and acquired cisplatin resistance in human ovarian cancer cells

While multiple changes are frequently found to be associated with cisplatin resistance in a variety of tumor cell lines, a cause-effect relationship of these alterations with the resistant phenotype has not been established. In order to identify the resistance-relevant determinants, a series of cisplatinresistant sublines with different degrees of resistance to cisplatin was developed in a human ovarian carcinoma cell line (O-129). Three derived resistant cell lines displayed 2.1-fold (O-129/DDP₄, low), 4.1-fold (O-129/DDP₈, moderate) and 6.3-fold (O-129/DDP₁₆, high) resistance, respectively, to cisplatin, compared with the sensitive parental line O-129. While the activity of poly(ADP-ribose) polymerase, an enzyme proposed to be involved in DNA repair, was elevated in all three resistant lines, a significant karyotypic change was observed only in the high-resistance line with the karyotype alteration from near diploidy to heteroploidy. The moderate (4.1-fold) and high (6.3-fold) DDP resistance was associated with a slow proliferation rate in drug-free medium, but cellular glutathione level was highly correlated with DDP sensitivity in all four cell lines. Taken together, the present studies establish that while many changes at cellular level can occur with development of cisplatin resistance, only elevation of intracellular glutathione concentration appears to be related to the resistance phenotype in these human ovarian cancer cells.Abbreviations DDP cisplatin - FBS fetal bovine serum - GSH glutathione - IC₅₀ drug concentration required to result in 50% growth inhibition - PARP poly(ADP-ribose) polymerase     

Determinants of cisplatin sensitivity in non-malignant non-drug-selected human T cell lines.

We have studied molecular mechanisms of cisplatin sensitivity and resistance in 3 non-malignant, non-drug-selected human T lymphocyte cell lines. HuT 78, H9, and MOLT-4 cells were assessed for sensitivity to cisplatin, DNA damage levels following defined drug exposures, drug accumulation, and DNA repair efficiency as measured by adduct removal from cellular DNA and by host-cell reactivation of cisplatin-modified plasmid DNA. Based on 3-day continuous drug exposures, the IC50 values for the cell lines were: HuT 78, 0.83 microM; H9, 0.45 microM; and MOLT-4, 0.33 microM. These cells retained this order with respect to DNA repair capability, whether measured by platinum-DNA adduct removal from cellular DNA or by host-cell reactivation assays. DNA repair values measured by these two assays were directly related to one another with a linear correlation coefficient of 0.993. At sublethal cisplatin doses the more resistant cells showed the highest levels of drug uptake. When drug uptake levels were 'corrected' for drug-induced cell kill, there were equal levels of DNA repair efficiency for a given level of drug uptake. Absolute levels of cisplatin-DNA adduct repair increased with increasing drug dose. However, at supralethal doses of drug, efficient DNA repair could be overcome in all 3 cell lines with percentage-adduct-removal dropping from a 60-80% range to a less than 30% range. We conclude that in non-malignant non-drug-selected human T cells, DNA repair appears to be the primary determinant of cisplatin sensitivity/resistance and that enhanced DNA repair may be a biologic compensatory mechanism for cells that cannot prevent cellular uptake of DNA-damaging agents.     

Inherent and Acquired Resistance to Paclitaxel in Hepatocellular Carcinoma: Molecular Events Involved

Hepatocellular carcinoma (HCC) is a primary malignancy of the liver and is a major cause of cancer related deaths worldwide. Only 10 to 20% of HCC can be surgically excised. Therefore, chemotherapeutic intervention and treatment is essential for achieving favorable prognosis. However, therapeutic outcome of chemotherapy is generally poor owing to inherent resistance of cancer cells to the treatment or due to development of acquired resistance. To differentiate and delineate the molecular events, we developed drug resistant Hep3B cells (DRC) by treating cells with the increasing concentration of paclitaxel. We also developed a unique single cell clone of Hep3B cells (SCC) by selecting single cell colonies and screening them for resistant phenotype. Interestingly, both DRC and SCC were resistant to paclitaxel in comparison to parental Hep3B cells. We analyzed the contributory factors that may be involved in the development of resistance. As expected, level of P-glycoprotein (P-gp) was elevated in DRC. In addition, Caveolin-1 (Cav-1), Fatty acid synthase (FASN) and Cytochrome P450 (CYP450) protein levels were elevated in DRC whereas in SCC, FASN and CYP450 levels were elevated. Downregulation of these molecules by respective siRNAs and/or by specific pharmacological inhibitors resensitized cells to paclitaxel. Interestingly, these drug resistant cells were also less sensitive to vinblastine, doxorubicin and methotrexate with the exception of cisplatin. Our results suggested that differential levels of P-gp, Cav-1 and FASN play a major role in acquired resistant phenotype whereas FASN level was associated with the presentation of inherent resistant phenotype in HCC.     

The induction of ferroptosis by impairing STAT3/Nrf2/GPx4 signaling enhances the sensitivity of osteosarcoma cells to cisplatin

Recent studies have indicated that promoting ferroptosis is a promising approach to attenuate drug resistance of cancer cells. Hence, this study aimed to induce ferroptosis in osteosarcoma cells, thereby increasing the sensitivity to cisplatin. Osteosarcoma cells MG63 and Saos‐2 were incubated with increasing doses of cisplatin to generate cisplatin‐resistant strains, MG63/DDP and Saos‐2/DDP. 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) and flow cytometry assays were performed to evaluate cell proliferation and cell death, respectively. Malondialdehyde (MDA), reactive oxygen species (ROS), and lipid oxidation in cells were measured to evaluate the degree of cell ferroptosis. MG63/DDP and Saos‐2/DDP cells showed increased viability and decreased death rate compared with MG63 and Saos‐2 cells, respectively, upon cisplatin treatment. Western blotting analysis indicated that protein levels of p‐STAT3 (Ser727), nuclear factor erythroid 2‐related factor 2 (Nrf2), and glutathione peroxidase 4 (GPx4) in drug‐resistant strains increased significantly in response to cisplatin. Co‐treatment with cisplatin and agonists of ferroptosis, Erastin, and RSL3, remarkably increased MDA, ROS, lipid oxidation, and sensitivity to cisplatin, in MG63/DDP and Saos‐2/DDP cells. Similar results were observed by co‐treatment of cells with cisplatin and a STAT3 inhibitor. The reduction of protein levels of p‐STAT3 (Ser727), Nrf2, and GPx4 in MG63/DDP and Saos‐2/DDP cells resulted in increased ferroptosis and sensitivity to cisplatin. These results indicate that cisplatin‐resistant osteosarcoma cells inhibited ferroptosis after exposure to low doses of cisplatin. However, ferroptosis agonists and STAT3 inhibitor reactivated ferroptosis in the cells and consequently increased sensitivity to cisplatin. This study demonstrates a new approach to attenuate resistance of osteosarcoma to cisplatin in vitro .     

miR-17-5p Downregulation Contributes to Paclitaxel Resistance of Lung Cancer Cells through Altering Beclin1 Expression

Non- small- cell lung cancer (NSCLC) is one of the most leading causes of cancer-related deaths worldwide. Paclitaxel based combination therapies have long been used as a standard treatment in aggressive NSCLCs. But paclitaxel resistance has emerged as a major clinical problem in combating non-small-cell lung cancer and autophagy is one of the important mechanisms involved in this phenomenon. In this study, we used microRNA (miRNA) arrays to screen differentially expressed miRNAs between paclitaxel sensitive lung cancer cells A549 and its paclitaxel-resistant cell variant (A549-T24). We identified miR-17-5p was one of most significantly downregulated miRNAs in paclitaxel-resistant lung cancer cells compared to paclitaxel sensitive parental cells. We found that overexpression of miR-17-5p sensitized paclitaxel resistant lung cancer cells to paclitaxel induced apoptotic cell death. Moreover, in this report we demonstrated that miR-17-5p directly binds to the 3′-UTR of beclin 1 gene, one of the most important autophagy modulator. Overexpression of miR-17-5p into paclitaxel resistant lung cancer cells reduced beclin1 expression and a concordant decease in cellular autophagy. We also observed similar results in another paclitaxel resistant lung adenosquamous carcinoma cells (H596-TxR). Our results indicated that paclitaxel resistance of lung cancer is associated with downregulation of miR-17-5p expression which might cause upregulation of BECN1 expression.     

Mechanisms of resistance to cisplatin

The use of cisplatin in cancer chemotherapy is limited by acquired or intrinsic resistance of cells to the drug. Cisplatin enters the cells and its chloride ligands are replaced by water, forming aquated species that react with nucleophilic sites in cellular macromolecules. The presence of the cisplatin adducts in DNA is thought to trigger cell cycle arrest and apoptosis. Knowledge of the mechanism of action of cisplatin has improved our understanding of resistance. Decreased intracellular concentration due to decreased drug uptake, increased reflux or increased inactivation by sulfhydryl molecules such as glutathione can cause resistance to cisplatin. Increased excision of the adducts from DNA by repair pathways or increased lesion bypass can also result in resistance. Finally, altered expression of regulatory proteins involved in signal transduction pathways that control the apoptotic pathway can also affect sensitivity to the drug. An improved understanding of the mechanisms of resistance operative in vivo has identified targets for intervention and may increase the utility of cisplatin for the treatment of cancer.     

Long-term cisplatin exposure impairs autophagy and causes cisplatin resistance in human lung cancer cells

Cisplatin-based chemotherapy frequently resulted in acquired resistance of cancer cells. The underlying mechanism of such resistance is not fully understood especially the involvement of autophagy and autophagic cell death. This study thus investigated whether an alteration in autophagy could be responsible for cisplatin resistance in the long-term exposure lung carcinoma cells. The cisplatin resistant clone (H460/cis) of H460 cells was established by exposing the cells with gradually increasing concentrations of cisplatin until chemoresistance acquisition was elucidated by MTT, Hoechst 33342 staining and comet assays. Degree of autophagosome formation and level of LC3 marker were evaluated by acridine orange and western blot analysis, respectively. H460/cis cells exhibited irregular shape with ~3-fold resistant to cisplatin-induced apoptosis compared with H460 cells. Proteins analysis for LC3 indicated that the levels of LC3 in resistant cells were significantly lower than those in H460 cells. Moreover, autophagosome formation detected by acridine orange staining was dramatically reduced in the resistant cells, suggesting the role of autophagy in attenuating of cisplatin-induced cell death. Further, co-treatment of cisplatin with autophagy inducer, trifluorperazine, could resensitize H460/cis cells to cisplatin-induced cell death. Our findings reveal the novel mechanisms causing cisplatin resistance in lung carcinoma cells after long-term drug exposure regarding autophagy.     

Modulation of mitomycin C resistance by glutathione transferase inhibitor ethacrynic acid.

This study was undertaken to elucidate the mechanism(s) of cross-resistance (4.9-fold) to mitomycin C (MMC) in a multi-drug-resistant cell line, P388/R-84. Intracellular accumulation of MMC by sensitive (P388/S) and P388/R-84 cells was comparable. Despite a 32% reduction in NADPH cytochrome P-450 reductase activity (responsible for MMC activation) in P388/R-84 cells, the rate of MMC bio-reduction by sensitive and resistant cells was similar. These results suggested that MMC resistance in P388/R-84 cell line must depend on factors other than impaired drug accumulation or bio-activation. Recent studies suggest that glutathione transferase (GST) dependent drug detoxification also contributes to cellular resistance of a variety of alkylating agents. Even though overexpression of GST has been noted in some MMC resistant tumor cells, it is not known if its level affects sensitivity to MMC. We have, therefore, determined the effect of ethacrynic acid (an inhibitor of GST activity) treatment on MMC cytotoxicity in P388/R-84 cells, which have about 2-fold higher GST activity than P388/S cells. The IC50 value for the inhibition of GST activity in vitro by ethacrynic acid (EA) was 16.5 microM (5 micrograms/ml). A depletion in intracellular GSH was also observed by treating P388/R-84 cells with EA alone or in combination with MMC. A non-toxic concentration of EA (1 microgram/ml; 3.3 microM) increased MMC cytotoxicity by 36% in P388/R-84 cells. MMC cytotoxicity was increased 2-fold by EA treatment in glutathione (GSH)-depleted P388/R-84 cells. These results suggest that GST mediated drug inactivation may represent another important mechanism of MMC resistance.     

Increased gene-specific repair of cisplatin interstrand cross-links in cisplatin-resistant human ovarian cancer cell lines.

We have studied several aspects of DNA damage formation and repair in human ovarian cancer cell lines which have become resistant to cisplatin through continued exposure to the anticancer drug. The resistant cell lines A2780/cp70 and 2008/c13*5.25 were compared with their respective parental cell lines, A2780 and 2008. Cells in culture were treated with cisplatin, and the two main DNA lesions formed, intrastrand adducts and interstrand cross-links, were quantitated before and after repair incubation. This quantitation was done for total genomic lesions and at the level of individual genes. In the overall genome, the initial frequency of both cisplatin lesions assayed was higher in the parental than in the derivative resistant cell lines. Nonetheless, the total genomic repair of each of these lesions was not increased in the resistant cells. These differences in initial lesion frequency between parental and resistant cell lines were not observed at the gene level. Resistant and parental cells had similar initial frequencies of intrastrand adducts and interstrand cross-links in the dihydrofolate reductase (DHFR) gene and in several other genes after cisplatin treatment of the cells. There was no increase in the repair efficiency of intrastrand adducts in the DHFR gene in resistant cell lines compared with the parental partners. However, a marked and consistent repair difference between parental and resistant cells was observed for the gene-specific repair of cisplatin interstrand cross-links. DNA interstrand cross-links were removed from three genes, the DHFR, multidrug resistance (MDR1), and delta-globin genes, much more efficiently in the resistant cell lines than in the parental cell lines. Our findings suggest that acquired cellular resistance to cisplatin may be associated with increased gene-specific DNA repair efficiency of a specific lesion, the interstrand cross-link.     

Generation and Characterisation of Cisplatin-Resistant Non-Small Cell Lung Cancer Cell Lines Displaying a Stem-Like Signature

Introduction

Inherent and acquired cisplatin resistance reduces the effectiveness of this agent in the management of non-small cell lung cancer (NSCLC). Understanding the molecular mechanisms underlying this process may result in the development of novel agents to enhance the sensitivity of cisplatin.

Methods

An isogenic model of cisplatin resistance was generated in a panel of NSCLC cell lines (A549, SKMES-1, MOR, H460). Over a period of twelve months, cisplatin resistant (CisR) cell lines were derived from original, age-matched parent cells (PT) and subsequently characterized. Proliferation (MTT) and clonogenic survival assays (crystal violet) were carried out between PT and CisR cells. Cellular response to cisplatin-induced apoptosis and cell cycle distribution were examined by FACS analysis. A panel of cancer stem cell and pluripotent markers was examined in addition to the EMT proteins, c-Met and β-catenin. Cisplatin-DNA adduct formation, DNA damage (γH2AX) and cellular platinum uptake (ICP-MS) was also assessed.

Results

Characterisation studies demonstrated a decreased proliferative capacity of lung tumour cells in response to cisplatin, increased resistance to cisplatin-induced cell death, accumulation of resistant cells in the G0/G1 phase of the cell cycle and enhanced clonogenic survival ability. Moreover, resistant cells displayed a putative stem-like signature with increased expression of CD133+/CD44+cells and increased ALDH activity relative to their corresponding parental cells. The stem cell markers, Nanog, Oct-4 and SOX-2, were significantly upregulated as were the EMT markers, c-Met and β-catenin. While resistant sublines demonstrated decreased uptake of cisplatin in response to treatment, reduced cisplatin-GpG DNA adduct formation and significantly decreased γH2AX foci were observed compared to parental cell lines.

Conclusion

Our results identified cisplatin resistant subpopulations of NSCLC cells with a putative stem-like signature, providing a further understanding of the cellular events associated with the cisplatin resistance phenotype in lung cancer.     

RING finger protein 38 induces the drug resistance of cisplatin in non-small-cell lung cancer

Cisplatin resistance of non-small-cell lung cancer (NSCLC) needs to be well elucidated. RING finger protein (RNF38) has been proposed as a biomarker of NSCLC poor prognosis. However, its role in drug resistance in NSCLC is poorly understood. RNF38 expression was detected in normal lung epithelial cell and four NSCLC cell lines. RNF38 was stably overexpressed in A549 and H460 cells or silenced in H1975 and cisplatin-resistant A549 cells (A549-CDDP resistant) using lentiviral vectors. RNF38 expression levels were determined using quantitative real-time polymerase chain reaction and western blotting analysis. Cell viability in response to different concentrations of cisplatin was evaluated by Cell Counting Kit-8 assay. RNF38 expression levels were markedly elevated in NSCLC cells and cells harboring high RNF38 were less sensitive to cisplatin. Overexpression of RNF38 reduced, while RNF38 silencing increased the drug sensitivity of cisplatin in NSCLC cells. Cisplatin-resistant cells expressed high RNF38 level. RNF38 silencing promoted cell apoptosis and enhanced the drug sensitivity of cisplatin in cisplatin-resistant NSCLC cells. These findings indicate that RNF38 might induce cisplatin resistance of NSCLC cells via promoting cell apoptosis and RNF38 could be a novel target for rectify cisplatin resistance in NSCLC cases.