Chronic cisplatin treatment promotes enhanced damage repair and tumor progression in a mouse model of lung cancer.

Chemotherapy resistance is a major obstacle in cancer treatment, yet the mechanisms of response to specific therapies have been largely unexplored in vivo. Employing genetic, genomic, and imaging approaches, we examined the dynamics of response to a mainstay chemotherapeutic, cisplatin, in multiple mouse models of human non-small-cell lung cancer (NSCLC). We show that lung tumors initially respond to cisplatin by sensing DNA damage, undergoing cell cycle arrest, and inducing apoptosis-leading to a significant reduction in tumor burden. Importantly, we demonstrate that this response does not depend on the tumor suppressor p53 or its transcriptional target, p21. Prolonged cisplatin treatment promotes the emergence of resistant tumors with enhanced repair capacity that are cross-resistant to platinum analogs, exhibit advanced histopathology, and possess an increased frequency of genomic alterations. Cisplatin-resistant tumors express elevated levels of multiple DNA damage repair and cell cycle arrest-related genes, including p53-inducible protein with a death domain (Pidd). We demonstrate a novel role for PIDD as a regulator of chemotherapy response in human lung tumor cells.

Multifaceted Mechanisms of Cisplatin Resistance in Long-Term Treated Urothelial Carcinoma Cell Lines.

Therapeutic efficacy of cisplatin-based treatment of late stage urothelial carcinoma (UC) is limited by chemoresistance. To elucidate underlying mechanisms and to develop new approaches for overcoming resistance, we generated long-term cisplatin treated (LTT) UC cell lines, characterised their cisplatin response, and determined the expression of molecules involved in cisplatin transport and detoxification, DNA repair, and apoptosis. Inhibitors of metallothioneins and Survivin were applied to investigate their ability to sensitise towards cisplatin. Cell growth, proliferation, and clonogenicity were examined after cisplatin treatment by MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, EdU (5-ethynyl-2'-deoxyuridine) incorporation assay, and Giemsa staining, respectively. Cell cycle distribution and apoptosis were quantified by flow cytometry. mRNA and protein expressions were measured by real-time quantitative (qRT)-PCR, western blot, or immunofluorescence staining. LTTs recovered rapidly from cisplatin stress compared to parental cells. In LTTs, to various extents, cisplatin exporters and metallothioneins were induced, cisplatin adduct levels and DNA damage were decreased, whereas expression of DNA repair factors and specific anti-apoptotic factors was elevated. Pharmacological inhibition of Survivin, but not of metallothioneins, sensitised LTTs to cisplatin, in an additive manner. LTTs minimise cisplatin-induced DNA damage and evade apoptosis by increased expression of anti-apoptotic factors. The observed diversity among the four LTTs highlights the complexity of cisplatin resistance mechanisms even within one tumour entity, explaining heterogeneity in patient responses to chemotherapy.

HIPEC ROC I: a phase I study of cisplatin administered as hyperthermic intraoperative intraperitoneal chemoperfusion followed by postoperative intravenous platinum-based chemotherapy in patients with platinum-sensitive recurrent epithelial ovarian cancer.

This phase I study tested the safety, feasibility, pharmacokinetics and pharmacodynamics of cisplatin administered as hyperthermic intraoperative intraperitoneal chemoperfusion (HIPEC) in patients with platinum-sensitive recurrent epithelial ovarian cancer (EOC) undergoing secondary cytoreductive surgery followed by postoperative platinum-based intravenous chemotherapy. Twelve patients with operable, recurrent platinum-sensitive EOC (recurrence ≥6 months after first-line therapy) were included according to the classical 3+3 dose-escalation design at three dose levels-60, 80 and 100 mg/m(2). After surgical cytoreduction, a single dose of cisplatin was administered via HIPEC for 90 min at 41-43°C. Postoperatively, all patients were treated with standard intravenous platinum-based combination chemotherapy. One of six patients experienced a dose-limiting toxicity (grade 3 renal toxicity) at a dose of 100 mg/m(2). The remaining five patients treated with 100 mg/m(2) tolerated their treatment well. The recommended phase II dose was established at 100 mg/m(2). The mean peritoneal-to-plasma AUC ratio was 19·5 at the highest dose level. Cisplatin-induced DNA adducts were confirmed in tumor samples. Common postoperative grade 1-3 toxicities included fatigue, postoperative pain, nausea, and surgical site infection. The ability to administer standard intravenous platinum-based chemotherapy after HIPEC was uncompromised. Cisplatin administered as HIPEC at a dose of 100 mg/m(2) has an acceptable safety profile in selected patients undergoing secondary cytoreductive surgery for platinum-sensitive recurrent EOC. Favorable pharmacokinetic and pharmacodynamic properties of HIPEC with cisplatin were confirmed at all dose levels, especially at 100 mg/m(2). The results are encouraging to determine the efficacy of HIPEC as a complementary treatment in patients with EOC.

Adaptation to Chronic-Cycling Hypoxia Renders Cancer Cells Resistant to MTH1-Inhibitor Treatment Which Can Be Counteracted by Glutathione Depletion.

Tumor hypoxia and hypoxic adaptation of cancer cells represent major barriers to successful cancer treatment. We revealed that improved antioxidant capacity contributes to increased radioresistance of cancer cells with tolerance to chronic-cycling severe hypoxia/reoxygenation stress. We hypothesized, that the improved tolerance to oxidative stress will increase the ability of cancer cells to cope with ROS-induced damage to free deoxy-nucleotides (dNTPs) required for DNA replication and may thus contribute to acquired resistance of cancer cells in advanced tumors to antineoplastic agents inhibiting the nucleotide-sanitizing enzyme MutT Homologue-1 (MTH1), ionizing radiation (IR) or both. Therefore, we aimed to explore potential differences in the sensitivity of cancer cells exposed to acute and chronic-cycling hypoxia/reoxygenation stress to the clinically relevant MTH1-inhibitor TH1579 (Karonudib) and to test whether a multi-targeting approach combining the glutathione withdrawer piperlongumine (PLN) and TH1579 may be suited to increase cancer cell sensitivity to TH1579 alone and in combination with IR. Combination of TH1579 treatment with radiotherapy (RT) led to radiosensitization but was not able to counteract increased radioresistance induced by adaptation to chronic-cycling hypoxia/reoxygenation stress. Disruption of redox homeostasis using PLN sensitized anoxia-tolerant cancer cells to MTH1 inhibition by TH1579 under both normoxic and acute hypoxic treatment conditions. Thus, we uncover a glutathione-driven compensatory resistance mechanism towards MTH1-inhibition in form of increased antioxidant capacity as a consequence of microenvironmental or therapeutic stress.

Transcriptional regulation of renal cytoprotective genes by Nrf2 and its potential use as a therapeutic target to mitigate cisplatin-induced nephrotoxicity.

The use of the chemotherapeutic drug cisplatin is limited in part by nephrotoxicity. Cisplatin causes renal DNA adducts and oxidative stress in rodents. The transcription factor Nrf2 (nuclear factor E2-related factor 2) induces expression of cytoprotective genes, including Nqo1 (NADPH:quinone oxidoreductase 1), Ho-1 (heme oxygenase-1), and Gclc (glutamate cysteine ligase catalytic subunit), in response to electrophilic and oxidative stress. In the present study, plasma and kidneys from wild-type and Nrf2-null mice were collected after receiving cisplatin for evaluation of renal injury, inflammation, mRNA, and protein expression. Compared with wild types, more extensive nephrotoxicity was observed in Nrf2-null mice after cisplatin treatment. Kidneys from Nrf2-null mice treated with cisplatin had more neutrophil infiltration accompanied by increased p65 nuclear factor κB binding and elevated inflammatory mediator mRNA levels. Cisplatin increased renal mRNA and protein expression of cytoprotective genes (Nqo1, Ho-1, Gclc) and transporters Mrp2 and Mrp4 in wild-type but not in Nrf2-null mice. Lastly, the Nrf2 activator, CDDO-Im [2-cyano-3,12-dioxooleana-1,9-dien-28-oic imidazolide], increased Nrf2 signaling in kidneys from wild-type mice and protected them from cisplatin toxicity. Collectively, these data indicate that the absence of Nrf2 exacerbates cisplatin renal damage and that pharmacological activation of Nrf2 may represent a novel therapy to prevent kidney injury. Coordinated regulation of detoxification enzymes and drug transporters and suppression of inflammation by Nrf2 during cisplatin nephrotoxicity are probable defense mechanisms to eliminate toxic mediators and promote proximal tubule recovery.

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

[This corrects the article DOI: 10.1371/journal.pone.0054193.].

Erythropoietin overrides the triggering effect of DNA platination products in a mouse model of cisplatin-induced neuropathy.

BACKGROUND: Cisplatin mediates its antineoplastic activity by formation of distinct DNA intrastrand cross links. The clinical efficacy and desirable dose escalations of cisplatin are restricted by the accumulation of DNA lesions in dorsal root ganglion (DRG) cells leading to sensory polyneuropathy (PNP). We investigated in a mouse model by which mechanism recombinant erythropoietin (rhEPO) protects the peripheral nervous system from structural and functional damage caused by cisplatin treatment with special emphasis on DNA damage burden. RESULTS: A cumulative dose of 16 mg cisplatin/kg resulted in clear electrophysiological signs of neuropathy, which were significantly attenuated by concomitant erythropoietin (cisplatin 32,48 m/s +/- 1,68 m/s; cisplatin + rhEPO 49,66 m/s +/- 1,26 m/s; control 55,01 m/s +/- 1,88 m/s; p < 0,001). The co-application of rhEPO, however, did not alter the level of unrepaired cisplatin-DNA lesions accumulating in DRG target cells. Micro-morphological analyses of the sciatic nerve from cisplatin-exposed mice showed damaged myelin sheaths and mitochondria. Co-administered rhEPO inhibited myelin sheaths from structural injuries and resulted in an increased number of intact mitochondria. CONCLUSION: The protective effect of recombinant erythropoietin is not mediated by reducing the burden of DNA platination in the target cells, but it is likely to be due to a higher resistance of the target cells to the adverse effect of DNA damage. The increased frequency of intact mitochondria might also contribute to this protective role.

Repair capacity for platinum-DNA adducts determines the severity of cisplatin-induced peripheral neuropathy.

The pronounced neurotoxicity of the potent antitumor drug cisplatin frequently results in the onset of peripheral polyneuropathy (PNP), which is assumed to be initially triggered by platination products in the nuclear DNA of affected tissues. To further elucidate the molecular mechanisms, we analyzed in a mouse model the formation and processing of the main cisplatin-induced DNA adduct (guanine-guanine intrastrand cross-link) in distinct neuronal cell types by adduct-specific monoclonal antibodies. Comparison of the adduct kinetics in cisplatin-injected mice either proficient or deficient for nucleotide excision repair (NER) functions revealed the essential role of this DNA repair pathway in protecting differentiated cells of the nervous system from excessive formation of such lesions. Hence, chronic exposure to cisplatin resulted in an accelerated accumulation of unrepaired intrastrand cross-links in neuronal cells of mice with dysfunctional NER. The augmented adduct levels in dorsal root ganglion (DRG) cells of those animals coincided with an earlier onset of PNP-like functional disturbance of their sensory nervous system. Independently from the respective repair phenotype, the amount of persisting DNA cross-links in DRG neurons at a given cumulative dose was significantly correlated to the degree of sensory impairment as measured by electroneurography. Collectively, these findings suggest a new model for the processing of cisplatin adducts in primary neuronal cells and accentuate the crucial role of effectual DNA repair capacity in the target cells for the individual risk of therapy-induced PNP.

DNA Ligases I and III Support Nucleotide Excision Repair in DT40 Cells with Similar Efficiency.

In eukaryotic cells helix-distorting DNA lesions like cyclobutane pyrimidine dimers (CPDs) and 6-4 pyrimidine-pyrimidone photoproducts (6-4 PPs) are efficiently removed by nucleotide excision repair (NER). NER is a multistep process where in the end, subsequent to replication over the gap, the remaining nick is sealed by a DNA ligase. Lig1 has been implicated as the major DNA ligase in NER. Recently, Lig3 has been implicated as a component of a NER subpathway that operates in dividing cells, but which becomes particularly important in nondividing cells. Here, we use DT40 cells and powerful gene targeting approaches for generating DNA ligase mutants to examine the involvement and contribution of Lig1 and Lig3 in NER using cell survival measured by colony formation, and repair kinetics of CPD by immunofluorescence microscopy and immuno-slot-blotting. Our results demonstrate an impressive and previously undocumented potential of Lig3 to substitute for Lig1 in removing helix-distorting DNA lesions by NER in proliferating cells. We show for the first time in a clean genetic background a functional redundancy in NER between Lig1 and Lig3, which appears to be cell cycle independent and which is likely to contribute to the stability of vertebrate genomes.

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 ß-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 ß-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.

Cisplatin resistance induced in germ cell tumour cells is due to reduced susceptibility towards cell death but not to altered DNA damage induction or repair.

To identify factors involved in cisplatin (CDDP) resistance of germ cell tumours (GCTs), we exposed NTERA-2 cells, and the platinum-adapted subline NTERA-2R to CDDP and compared their response. While both cell lines showed comparable proliferation, NTERA-2R cells were clearly more resistant to the drug than the parental NTERA-2 cell line. Interestingly, the two lines showed identical extent of DNA adduct formation and elimination, indicating that neither changes in CDDP uptake, nor altered drug efflux, DNA binding, or repair caused the difference in resistance. Similarly, no difference occurred in the time-course of γH2AX formation, which was not linked to 53BP1 accumulation. In contrast, NTERA-2R cells showed a more pronounced dose-dependent S phase delay, a transient G(2)/M-block, and subsequent release into immediate cell death. We thus conclude that the enhanced resistance against CDDP is linked to reduced susceptibility to cell death rather than to an altered DNA adduct formation or adduct removal.

Recombinant human erythropoietin counteracts cisplatin-induced visceral hyperalgesia.

OBJECTIVE: Cisplatin exerts its cytotoxic effect through distinct DNA lesions, leading to peripheral neuropathy. The risk of sensory neuropathy is a common problem during cancer treatment with cisplatin, leading to somatic hyperalgesia. Yet, data focussing on cisplatin-induced impairment of the autonomic nervous system are limited. The present study was aimed to investigate the effect of recombinant human erythropoietin (rhEPO) on cisplatin-induced visceral hyperalgesia. METHODS: C57BL/6 mice were treated either with cisplatin (2 mg/kg, once per week) or with cisplatin (2 mg/kg, once per week) plus rhEPO (40 microg/kg, 3 times per week) for 8 weeks. Controls were treated with saline. To quantify the visceromotor response (VMR) at week 9, standardized electrodes were implanted into the external oblique musculature for electromyographic recordings. After that, animals were decapitated and dorsal root ganglia (DRG) was removed for transmission electron microscopy studies. RESULTS: Cisplatin-treated mice showed a significant increase of VMR compared to the controls [(7080 +/- 969) vs (2864 +/- 279); P< 0.001], while rhEPO dramatically counteracted this effect [(2962 +/- 336) vs (7080 +/- 969); P< 0.001)]. Transmission electron microscopy revealed cisplatin-induced structural lesions of nuclear membrane in DRG cells, which could be ameliorated by rhEPO. CONCLUSION: Erythropoietin can significantly ameliorate the cisplatin-induced visceral hyperplasia and DRG nuclear membrane structure damage in mice, indicating a neuroprotective role of erythropoietin.

High accumulation of platinum-DNA adducts in strial marginal cells of the cochlea is an early event in cisplatin but not carboplatin ototoxicity.

Ototoxicity is a typical dose-limiting side effect of cancer chemotherapy with cisplatin but much less so with carboplatin. To elucidate the underlying molecular pathological mechanisms, we have measured the formation and persistence of drug-induced DNA adducts in the nuclei of inner ear cells of guinea pigs after short-term exposure to either cisplatin or carboplatin using immunofluorescence staining and quantitative image analysis. After application of carboplatin, all cells of the cochlea exhibited a similar burden of guanine-guanine intrastrand cross-links in DNA. In contrast, we observed a pronounced 3- to 5-fold accumulation of this cytotoxic adduct exclusively in the marginal cells of the stria vascularis between 8 and 48 h after treatment with cisplatin. In the kidney, the other critical target tissue of cisplatin toxicity, a similar high preferential formation of cytotoxic DNA adducts was measured in the tubular epithelial cells but not in other renal cell types. As for the ear, this excessive formation of DNA damage in a particular cell type was seen in animals treated with cisplatin but not those treated with carboplatin. Because cisplatin ototoxicity is often attributed to oxidative stress mediated by the generation of radical oxygen species (ROS), we have measured in parallel the levels of the lead DNA oxidation product 8-oxoguanine (8-oxoG) in cochlear cryosections. Compared with basal levels in untreated control cochleas, no additional formation of 8-oxoG was detectable up to 48 h after cisplatin treatment in the DNA of either inner-ear cell type. This suggests that the generation of ROS may be a secondary event in cisplatin ototoxicity.