Repair of lesions induced in human liver cell DNA by N-nitroso compounds as measured by the bromodeoxyuridine photolysis method

Chang human liver cells were treated with the carcinogens N-methyl-N′-nitrosoguanidine (MNNG) and nitrosomorpholine (NM). In addition, cells were exposed to the folic acid analog, 2-hydroxy-N¹⁰ nitrosofolic acid. Repair of the damage to DNA was estimated by selective photolysis of BUdR-containing repaired regions with 313 nm radiation. The influence of the co-carcinogen Arlacel A was estimated with the three compounds. Results indicated significant repair synthesis with MNNG- and NM-treated cells. 2-Hydroxy-N¹⁰ nitrosofolic acid elicited no damage to the liver DNA. Arlacel A prevented repair synthesis in cells treated with NM and MNNG.     

Proteasome inhibition prevents cell death induced by the chemotherapeutic agent cisplatin downstream of DNA damage

Cisplatin is a highly effective chemotherapeutic drug acting as a DNA-damaging agent that induces apoptosis of rapidly proliferating cells. Unfortunately, cellular resistance still occurs. Mutations in p53 in a large fraction of tumor cells contribute to defects in apoptotic pathways and drug resistance. To uncover new strategies to eliminate tumors through a p53-independent pathway, we established a simplified model devoid of p53 to study cisplatin-induced regulated cell death, using the yeast Saccharomyces cerevisiae. We previously showed that cisplatin induces an active form of cell death accompanied by DNA condensation and fragmentation/degradation, but no significant mitochondrial dysfunction. We further demonstrated that proteasome inhibition, either with MG132 or genetically, increased resistance to cisplatin. In this study, we sought to determine how proteasome inhibition is important for cisplatin resistance by analyzing how it affects several phenotypes associated with the DNA damage response. We found MG132 does not seem to affect the activation of the DNA damage response or increase damage tolerance. Moreover, central modulators of the DNA damage response are not required for cisplatin resistance imparted by MG132. These results suggest the proteasome is involved in modulation of cisplatin toxicity downstream of DNA damage. Proteasome inhibitors can sensitize tumor cells to cisplatin, but protect others from cisplatin-induced cell death. Elucidation of this mechanism will therefore aid in the development of new strategies to increase the efficacy of chemotherapy.     

Protein Expression of DNA Damage Repair Proteins Dictates Response to Topoisomerase and PARP Inhibitors in Triple-Negative Breast Cancer

Patients with metastatic triple-negative breast cancer (TNBC) have a poor prognosis. New approaches for the treatment of TNBC are needed to improve patient survival. The concept of synthetic lethality, brought about by inactivating complementary DNA repair pathways, has been proposed as a promising therapeutic option for these tumors. The TNBC tumor type has been associated with BRCA mutations, and inhibitors of Poly (ADP-ribose) polymerase (PARP), a family of proteins that facilitates DNA repair, have been shown to effectively kill BRCA defective tumors by preventing cells from repairing DNA damage, leading to a loss of cell viability and clonogenic survival. Here we present preclinical efficacy results of combining the PARP inhibitor, ABT-888, with CPT-11, a topoisomerase I inhibitor. CPT-11 binds to topoisomerase I at the replication fork, creating a bulky adduct that is recognized as damaged DNA. When DNA damage was stimulated with CPT-11, protein expression of the nucleotide excision repair enzyme ERCC1 inversely correlated with cell viability, but not clonogenic survival. However, 4 out of the 6 TNBC cells were synergistically responsive by cell viability and 5 out of the 6 TNBC cells were synergistically responsive by clonogenic survival to the combination of ABT-888 and CPT-11. In vivo, the BRCA mutant cell line MX-1 treated with CPT-11 alone demonstrated significant decreased tumor growth; this decrease was enhanced further with the addition of ABT-888. Decrease in tumor growth correlated with an increase in double strand DNA breaks as measured by γ-H2AX phosphorylation. In summary, inhibiting two arms of the DNA repair pathway simultaneously in TNBC cell lines, independent of BRCA mutation status, resulted in un-repairable DNA damage and subsequent cell death.     

Folate Deficiency Provides Protection against Colon Carcinogenesis in DNA Polymerase β Haploinsufficient Mice

Aging and DNA polymerase β deficiency (β-pol^+/−) interact to accelerate the development of malignant lymphomas and adenocarcinoma and increase tumor bearing load in mice. Folate deficiency (FD) has been shown to induce DNA damage repaired via the base excision repair (BER) pathway. We anticipated that FD and BER deficiency would interact to accelerate aberrant crypt foci (ACF) formation and tumor development in β-pol haploinsufficient animals. FD resulted in a significant increase in ACF formation in wild type (WT) animals exposed to 1,2-dimethylhydrazine, a known colon and liver carcinogen; however, FD reduced development of ACF in β-pol haploinsufficient mice. Prolonged feeding of the FD diet resulted in advanced ACF formation and liver tumors in wild type mice. However, FD attenuated onset and progression of ACF and prevented liver tumorigenesis in β-pol haploinsufficient mice, i.e. FD provided protection against tumorigenesis in a BER-deficient environment in all tissues where 1,2-dimethylhydrazine exerts its damage. Here we show a distinct down-regulation in DNA repair pathways, e.g. BER, nucleotide excision repair, and mismatch repair, and decline in cell proliferation, as well as an up-regulation in poly(ADP-ribose) polymerase, proapoptotic genes, and apoptosis in colons of FD β-pol haploinsufficient mice.     

Chemopreventive effect of bilberry (Vaccinium myrtillus) against cisplatin-induced oxidative stress and DNA damage as shown by the comet assay in peripheral blood of rats

The aim of this study was to evaluate the chemopreventive effect of bilberry on cisplatin induced oxidative stress and DNA damage in rat blood. Twenty-one female Wistar-Albino rats were divided into three groups: group I — untreated; group II — treated with cisplatin (single dose 7.5 mg/kg b.w.); and group III — treated with cisplatin (single dose 7.5 mg/kg b.w.) and bilberry (200 mg/kg b.w. for 10 days). Antioxidant enzyme systems including superoxide dismutase, catalase, glutathione peroxidase and the level of malondialdehyde (MDA) that might occur on erythrocytes have been determined and single cell gel electrophoresis (comet) was utilized to evaluate the DNA damage in lymphocytes. Treatment with cisplatin has increased the levels of MDA and decreased antioxidant enzymes in erythrocytes. Comet assay showed significantly higher values at dose of 7.5 mg/kg cisplatin as the result of oxidative DNA damage when compared to the control group. Cisplatin treatment with bilberry resulted in a highly significant (P < 0.05) decreased in the lymphocytes DNA when compared to the cisplatin group. Bilberry has been effective on antioxidant enzyme systems and MDA level and significantly decreased the comets. Our results indicate that bilberry is capable of preventing genotoxic and cytotoxic damage caused by cisplatin in peripheral blood cells in rats.     

Glutathione depletion sensitizes cisplatin- and temozolomide-resistant glioma cells in vitro and in vivo

Malignant glioma is a severe type of brain tumor with a poor prognosis and few options for therapy. The main chemotherapy protocol for this type of tumor is based on temozolomide (TMZ), albeit with limited success. Cisplatin is widely used to treat several types of tumor and, in association with TMZ, is also used to treat recurrent glioma. However, several mechanisms of cellular resistance to cisplatin restrict therapy efficiency. In that sense, enhanced DNA repair, high glutathione levels and functional p53 have a critical role on cisplatin resistance. In this work, we explored several mechanisms of cisplatin resistance in human glioma. We showed that cellular survival was independent of the p53 status of those cells. In addition, in a host-cell reactivation assay using cisplatin-treated plasmid, we did not detect any difference in DNA repair capacity. We demonstrated that cisplatin-treated U138MG cells suffered fewer DNA double-strand breaks and DNA platination. Interestingly, the resistant cells carried higher levels of intracellular glutathione. Thus, preincubation with the glutathione inhibitor buthionine sulfoximine (BSO) induced massive cell death, whereas N-acetyl cysteine, a precursor of glutathione synthesis, improved the resistance to cisplatin treatment. In addition, BSO sensitized glioma cells to TMZ alone or in combination with cisplatin. Furthermore, using an in vivo model the combination of BSO, cisplatin and TMZ activated the caspase 3–7 apoptotic pathway. Remarkably, the combined treatment did not lead to severe side effects, while causing a huge impact on tumor progression. In fact, we noted a remarkable threefold increase in survival rate compared with other treatment regimens. Thus, the intracellular glutathione concentration is a potential molecular marker for cisplatin resistance in glioma, and the use of glutathione inhibitors, such as BSO, in association with cisplatin and TMZ seems a promising approach for the therapy of such devastating tumors.Malignant gliomas are the most common and aggressive type of primary brain tumor in adults. Current therapy includes surgery for tumor resection, followed by radiotherapy and/or concomitant adjuvant chemotherapy with temozolomide (TMZ) or chloroethylating nitrosoureas (CNUs). However, these protocols have limited success, and patients diagnosed with glioma have a dismal prognosis, with a median survival of 15 months and a 5-year survival rate of ~2%.¹ Several molecular mechanisms for cell resistance to these agents have been described. Because both are alkylating agents, the repair enzyme O₆-methylguanine-DNA methyltransferase (MGMT) is certainly a first barrier that is associated with increased tumor resistance.^{2, 3} The p53 status has also been proposed to act in an opposite manner in glioma cell resistance to TMZ or CNUs. Although p53 mutation is shown to be more resistant to TMZ treatment, owing to the induction of cell death,⁴ the p53 protein protects glioma cells after CNU treatment, most likely by improving other DNA repair systems.⁵Cisplatin is one of the most effective anticancer drugs and is used as a first-line treatment for a wide spectrum of solid tumors, such as ovarian, lung and testicular cancer,⁶ and it is used for adjuvant therapy in gliomas.⁷ Cisplatin is a molecule formed by one platinum ion that is surrounded by four ligands at the cis position: two chloride atoms and two amine molecules. The mechanism of action of cisplatin is mainly based on DNA damage. Once inside the cell, cisplatin becomes activated by the substitution of one or two chloride atoms by water, a process known as aquation. Owing to this process, the drug becomes positively charged and interacts with the DNA molecule, inducing the formation of DNA adducts. Activated cisplatin preferentially binds to purine bases in the nucleophilic N7 sites, where the majority of adducts occur between two guanines on the same strand, whereas ~3–5% of cisplatin adducts react with purines at the opposite strands, forming interstrand crosslinks (ICLs). The DNA lesions, in turn, trigger a series of signal-transduction pathways, leading to cell-cycle arrest, DNA repair and apoptosis.⁸Although relatively efficient, resistance to cisplatin, either intrinsic or acquired, during cycles of therapy is common, and overcoming tumor resistance remains the major challenge for cisplatin anticancer therapy. Cellular cisplatin resistance is a multifactorial phenomenon that may include decreased drug uptake, enhanced DNA repair capacity and higher glutathione (GSH) concentration.⁹GSH is a highly abundant, low-molecular-weight peptide in the cell, and it is well known for its critical importance in maintaining the cellular oxidative balance as a free radical scavenger. Additionally, GSH has a protective role against xenobiotic agents once its highly reactive thiol group binds and inactivates those agents. In fact, the GSH content and glutathione S-transferase (GST) have long been associated with cisplatin resistance in numerous cell lines and tumor tissues.^{9, 10, 11}Considering these possible pathways, it is not clear, however, which one determinates cisplatin resistance in glioma cells. Aiming to better understand the molecular mechanisms of resistance to this drug, four human glioma cell lines with different p53 status were investigated. We showed that cellular resistance was found to be independent of p53 as well as of the DNA repair capacity of the cells. On the other hand, the GSH levels within the cell were shown to act as a decisive resistance barrier to cisplatin, reducing the induction of DNA damage in the treated cells. Also, both in an in vitro and in vivo model depletion of GSH by an inhibitor (buthionine sulfoximine, BSO) sensitized the glioma cell lines to cisplatin. Interestingly, BSO also potentiated TMZ cytotoxicity. Thus, combination with BSO, cisplatin and TMZ turned out to be an extremely powerful approach to improve cytotoxicity in glioma, thus providing an exciting alternative for glioma treatment.     

DNA damage-induced inhibition of rRNA synthesis by DNA-PK and PARP-1

RNA synthesis and DNA replication cease after DNA damage. We studied RNA synthesis using an in situ run-on assay and found ribosomal RNA (rRNA) synthesis was inhibited 24 h after UV light, gamma radiation or DNA cross-linking by cisplatin in human cells. Cisplatin led to accumulation of cells in S phase. Inhibition of the DNA repair proteins DNA-dependent protein kinase (DNA-PK) or poly(ADP-ribose) polymerase 1 (PARP-1) prevented the DNA damage-induced block of rRNA synthesis. However, DNA-PK and PARP-1 inhibition did not prevent the cisplatin-induced arrest of cell cycle in S phase, nor did it induce de novo BrdU incorporation. Loss of DNA-PK function prevented activation of PARP-1 and its recruitment to chromatin in damaged cells, suggesting regulation of PARP-1 by DNA-PK within a pathway of DNA repair. From these results, we propose a sequential activation of DNA-PK and PARP-1 in cells arrested in S phase by DNA damage causes the interruption of rRNA synthesis after DNA damage.     

Rhus verniciflua Stokes prevents cisplatin-induced cytotoxicity and reactive oxygen species production in MDCK-I renal cells and intact mice

Cisplatin-induced oxidative stress can cause liver and kidney damage, thus limiting therapeutic efficacy. Thus, in the present study, since Rhus verniciflua Stokes (RVS) containing flavonoids has antioxidant effects, we investigated whether it can protect cisplatin-induced toxicity in vitro and in vivo, The in vitro effects of RVS on the cell viability and reactive oxygen species (ROS) production were investigated using cisplatin-treated Madin–Darby Canine kidney (MDCK)-I renal cells. Its in vivo effects were also studied in BALB/c mice inoculated with CT-26 colon adenocarcinoma cells and treated with cisplatin with or without RVS. Liver and renal functions were assessed together with indices of tissue oxidation. RVS prevented cisplatin-induced cytotoxicity and ROS release against MDCK-I cells. RVS alone exerted modest antitumor activity against CT-26 cells. When used concurrently with cisplatin, RVS prevented the increases in serum blood urea nitrogen (BUN), creatinine, alanine aminotransferase (ALT), aspartate aminotransferase (AST), and NO, while reducing liver and kidney tissue MDA content, and increasing catalase, glutathione (GSH), and superoxide dismutase (SOD) activities. Moreover, the antitumor efficacy of cisplatin was not altered by concurrent administration of RVS. These findings demonstrate that RVS prevents cisplatin-induced toxicity in vitro and in vivo via an antioxidant activity without hurting its antitumor effectiveness, suggesting that RVS can be usefully applied to the neoplastic patients as a combined chemopreventive agent with cisplatin.     

Regulation and Functional Contribution of Thymidine Kinase 1 in Repair of DNA Damage

Cellular supply of dNTPs is essential in the DNA replication and repair processes. Here we investigated the regulation of thymidine kinase 1 (TK1) in response to DNA damage and found that genotoxic insults in tumor cells cause up-regulation and nuclear localization of TK1. During recovery from DNA damage, TK1 accumulates in p53-null cells due to a lack of mitotic proteolysis as these cells are arrested in the G₂ phase by checkpoint activation. We show that in p53-proficient cells, p21 expression in response to DNA damage prohibits G₁/S progression, resulting in a smaller G₂ fraction and less TK1 accumulation. Thus, the p53 status of tumor cells affects the level of TK1 after DNA damage through differential cell cycle control. Furthermore, it was shown that in HCT-116 p53^−/− cells, TK1 is dispensable for cell proliferation but crucial for dTTP supply during recovery from DNA damage, leading to better survival. Depletion of TK1 decreases the efficiency of DNA repair during recovery from DNA damage and generates more cell death. Altogether, our data suggest that more dTTP synthesis via TK1 take place after genotoxic insults in tumor cells, improving DNA repair during G₂ arrest.     

Targeted CUL4A inhibition synergizes with cisplatin to yield long-term survival in models of head and neck squamous cell carcinoma through a DDB2-mediated mechanism

Patients with late-stage and human papillomavirus (HPV)-negative head and neck squamous cell carcinoma (HNSCC) continue to have a very poor prognosis. The development of more effective novel therapies that improve overall survival and overcome drug resistance is an urgent priority. Here we report that HNSCC tumors significantly overexpress NEDD8 and exhibit high sensitivity to the first-in-class NEDD8-activating enzyme (NAE) inhibitor pevonedistat. Additional studies established that disruption of NEDD8-mediated protein turnover with pevonedistat dramatically augmented cisplatin-induced DNA damage and apoptosis in HNSCC models. Further analysis revealed that the specific pevonedistat target CUL4A played an essential role in driving the synergy of the pevonedistat and cisplatin combination. Targeted inhibition of CUL4A resulted in significant downregulation in Damage Specific DNA binding protein 2 (DDB2), a DNA-damage recognition protein that promotes nucleotide excision repair and resistance to cisplatin. Silencing of CUL4A or DDB2 enhanced cisplatin-induced DNA damage and apoptosis in a manner similar to that of pevonedistat demonstrating that targeted inhibition of CUL4A may be a novel approach to augment cisplatin therapy. Administration of pevonedistat to mice bearing HNSCC tumors significantly decreased DDB2 expression in tumor cells, increased DNA damage and potently enhanced the activity of cisplatin to yield tumor regression and long-term survival of all animals. Our findings provide strong rationale for clinical investigation of CUL4A inhibition with pevonedistat as a novel strategy to augment the efficacy of cisplatin therapy for patients with HNSCC and identify loss of DDB2 as a key pharmacodynamic mediator controlling sensitivity to this regimen.Subject terms: Preclinical research, Translational research, Cancer therapeutic resistance, Oral cancer     

Roles of ChlR1 DNA helicase in replication recovery from DNA damage

The ChlR1 DNA helicase is mutated in Warsaw breakage syndrome characterized by developmental anomalies, chromosomal breakage, and sister chromatid cohesion defects. However, the mechanism by which ChlR1 preserves genomic integrity is largely unknown. Here, we describe the roles of ChlR1 in DNA replication recovery. We show that ChlR1 depletion renders human cells highly sensitive to cisplatin; an interstrand-crosslinking agent that causes stalled replication forks. ChlR1 depletion also causes accumulation of DNA damage in response to cisplatin, leading to a significant delay in resolution of DNA damage. We also report that ChlR1-depleted cells display defects in the repair of double-strand breaks induced by the I-PpoI endonuclease and bleomycin. Furthermore, we demonstrate that ChlR1-depeleted cells show significant delays in replication recovery after cisplatin treatment. Taken together, our results indicate that ChlR1 plays an important role in efficient DNA repair during DNA replication, which may facilitate efficient establishment of sister chromatid cohesion.     

A comparison of in vitro platinum-DNA adduct formation between carboplatin and cisplatin

• 1.1. DNA damage induced by carboplatin [cis-diammine-(1,1-cyclobutanedi-carboxylato)platinum(II)] was studied in vitro in comparison with cisplatin [cis-diammine-dichloroplatinum(II)]. The drug-induced DNA damage monitored by conformational change of pUC18 plasmid DNA showed that carboplatin required 10 times higher drug concentration and 7.5 times longer incubation time than those of cisplatin to induce the same degree of conformational change on plasmid DNA.
• 2.2. The carboplatin-induced DNA damage was promoted by the increase of pH of the reaction mixture for platinum-DNA adduct formation.
• 3.3. Sequence gel analysis of carboplatin-damaged DNA indicated that carboplatin attacked preferentially the sequence of GG > AG > GA > GNG in the order, similarly to the case of cisplatin.
• 4.4. DNA adducts formed by carboplatin were analyzed by HPLC after a sequential digestion of carboplatin-treated DNA with deoxyribonuclease I and S1 nuclease. A single peak having the same retention time as that of bifunctional adduct of (dGMP)₂Pt(NH₃)₂ appeared by treating DNA with carboplatin. The adduct was assigned to be d(pGpG) > Pt(NH₃)₂.
• 5.5. These results suggested that carboplatin induces the same platinum-DNA adducts as those induced by cisplatin, and that the difference in efficiency or kinetics of DNA damage between carboplatin and cisplatin is due to difference of aquation rate between them.

     

Curcumin induces apoptosis and inhibits growth of orthotopic human non-small cell lung cancer xenografts

Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related mortality. Curcumin is involved in various biological pathways leading to inhibition of NSCLC growth. The purpose of this study was to evaluate the effect of curcumin on expression of nuclear factor κB-related proteins in vitro and in vivo and on growth and metastasis in an intralung tumor mouse model.H1975 NSCLC cells were treated with curcumin (0–50 μM) alone, or combined with gemcitabine or cisplatin. The effects of curcumin were evaluated in cell cultures and in vivo, using ectopic and orthotopic lung tumor mouse models. Twenty mice were randomly selected into two equal groups, one that received AIN-076 control diet and one that received the same food but with the addition of 0.6% curcumin 14 days prior to cell implantation and until the end of the experiment. To generate orthotopic tumor, lung cancer cells in Matrigel were injected percutaneously into the left lung of CD-1 nude mice. Western blot analysis showed that the expressions of IkB, nuclear p65, cyclooxygenase 2 (COX-2) and p-ERK1/2 were down-regulated by curcumin in vitro. Curcumin potentiated the gemcitabine- or cisplatin-mediated antitumor effects. Curcumin reduced COX-2 expression in subcutaneous tumors in vivo and caused a 36% decrease in weight of intralung tumors (P=.048) accompanied by a significant survival rate increase (hazard ratio=2.728, P=.036). Curcumin inhibition of COX-2, p65 expression and ERK1/2 activity in NSCLC cells was associated with decreased survival and increased induction of apoptosis. Curcumin significantly reduced tumor growth of orthotopic human NSCLC xenografts and increased survival of treated athymic mice. To evaluate the role of curcumin in chemoprevention and treatment of NSCLC, further clinical trials are required.     

Matched tissue and liquid biopsies for advanced non-small cell lung cancer patients A potentially indispensable complementary approach

The introduction of liquid biopsies (LB) has brought forth a number of therapeutic opportunities into the domain of thoracic oncology. Many of which have been adopted for care of patients presenting with advanced non-squamous non-small cell lung cancer (aNS-NSCLC). For example, one of the most frequent indications to perform a LB in these patients, at least in Europe, is for patients treated with tyrosine kinase inhibitors (TKIs) targeting EGFR and ALK genomic alterations when the tumor progresses. A tissue biopsy (TB) must then be taken, ideally from a site of a tumor that progresses, in particular if the LB does not permit detection of a mechanism of resistance to TKI. A LB from a patient with aNS-NSCLC is recommended before first-line therapy if no tissue and/or cytological material is accessible or if the extracted nucleic acid is insufficient in amount and/or of poor quality. At present a LB and a TB are rarely performed simultaneously before treatment and/or on tumor progression. This complementary/matched testing approach is still controversial but needs to be better evaluated to determine the true benefit to care of patients. This review provides an update on the complementarity of the LB and TB method for care of patients presenting with aNS-NSCLC.     

DNA-Bound Platinum Is the Major Determinant of Cisplatin Sensitivity in Head and Neck Squamous Carcinoma Cells

Purpose

The combination of systemic cisplatin with local and regional radiotherapy as primary treatment of head and neck squamous cell carcinoma (HNSCC) leads to cure in approximately half of the patients. The addition of cisplatin has significant effects on outcome, but despite extensive research the mechanism underlying cisplatin response is still not well understood.

Methods

We examined 19 HNSCC cell lines with variable cisplatin sensitivity. We determined the TP53 mutational status of each cell line and investigated the expression levels of 11 potentially relevant genes by quantitative real-time PCR. In addition, we measured cisplatin accumulation and retention, as well as the level of platinum-DNA adducts.

Results

We found that the IC₅₀ value was significantly correlated with the platinum-DNA adduct levels that accumulated during four hours of cisplatin incubation (p = 0.002). We could not find a significant correlation between cisplatin sensitivity and any of the other parameters tested, including the expression levels of established cisplatin influx and efflux transporters. Furthermore, adduct accumulation did not correlate with mRNA expression of the investigated influx pumps (CTR1 and OCT3) nor with that of the examined DNA repair genes (ATR, ATM, BRCA1, BRCA2 and ERCC1).

Conclusion

Our findings suggest that the cisplatin-DNA adduct level is the most important determinant of cisplatin sensitivity in HNSCC cells. Imaging with radio-labeled cisplatin might have major associations with outcome.     

A potent aminonaphthalimide platinum(IV) complex with effective antitumor activities in vitro and in vivo displaying dual DNA damage effects on tumor cells

A new aminonaphthalimide platinum(IV) complex was developed by incorporating aminonaphthalimide, a DNA intercalator, into the platinum(IV) system. This complex displayed potent antitumor activities against all tested tumor cell lines in vitro and showed great potential in overcoming drug resistance of cisplatin. Moreover, it remarkably inhibited the growth of CT26 xenografts in BALB/c mice without severe side effects in vivo. Then, the compound exhibited a dual DNA damage antitumor mechanism that it could interact with DNA in tetravalent form via the naphthalimide group to cause DNA lesion, and the further liberation of platinum(II) complex after reduction would induce remarkable secondary damage to DNA. Meanwhile, it caused cell apoptosis through an intrinsic apoptosis pathway by up-regulating the expression of caspase 3 and caspase 9.     

AtMMS21 regulates DNA damage response and homologous recombination repair in Arabidopsis

DNA damage is a significant problem in living organisms and DNA repair pathways have been evolved in different species to maintain genomic stability. Here we demonstrated the molecular function of AtMMS21, a component of SMC5/6 complex, in plant DNA damage response. Compared with wild type, the AtMMS21 mutant plants show hypersensitivity in the DNA damaging treatments by MMS, cisplatin and gamma radiation. However, mms21-1 is not sensitive to replication blocking agents hydroxyurea and aphidicolin. The expression of a DNA damage response gene PARP2 is upregulated in mms21-1 under normal condition, suggesting that this signaling pathway is constitutively activated in the mutant. Depletion of ATAXIA-TELANGIECTASIA MUTATED (ATM) in mms21-1 enhances its root growth defect phenotype, indicating that ATM and AtMMS21 may play additive roles in DNA damage pathway. The analysis of homologous recombination frequency showed that the number of recombination events is reduced in mms21-1 mutant. Conclusively, we provided evidence that AtMMS21 plays an important role in homologous recombination for DNA damage repair.     

Inhibition of double-strand break non-homologous end-joining by cisplatin adducts in human cell extracts

The effect of cis-diaminedichloroplatinum(II) (cisplatin) DNA damage on the repair of double-strand breaks by non-homologous end-joining (NHEJ) was determined using cell-free extracts. NHEJ was dramatically decreased when plasmid DNA was damaged to contain multiple types of DNA adducts, along the molecule and at the termini, by incubation of DNA with cisplatin; this was a cisplatin concentration-dependent effect. We investigated the effect a single GTG cisplatination site starting 10 bp from the DNA termini would have when surrounded by the regions of AT-rich DNA which were devoid of the major adduct target sequences. Cisplatination of a substrate containing short terminal 13–15 bp AT-rich sequences reduced NHEJ to a greater extent than that of a substrate with longer (31–33 bp) AT-rich sequences. However, cisplatination at the single GTG site within the AT sequence had no significant effect on NHEJ, owing to the influence of additional minor monoadduct and dinucleotide adduct sites within the AT-rich region and owing to the influence of cisplatination at sites upstream of the AT-rich regions. We then studied the effect on NHEJ of one cis-[Pt(NH₃)₂{d(GpTpG)-N7(1),-N7(3)} [abbreviated as 1,3-d(GpTpG)] cisplatin adduct in the entire DNA molecule, which is more reflective of the situation in vivo during concurrent chemoradiation. The presence of a single 1,3-d(GpTpG) cisplatin adduct 10 bases from each of the two DNA ends to be joined resulted in a small (30%) but significant decrease in NHEJ efficiency. This process, which was DNA-dependent protein kinase and Ku dependent, may in part explain the radiosensitizing effect of cisplatin administered during concurrent chemoradiation.     

Cisplatin resistance: Preclinical findings and clinical implications

Cisplatin is used for the treatment of many types of solid cancers. While testicular cancers respond remarkably well to cisplatin, the therapeutic efficacy of cisplatin for other solid cancers is limited because of intrinsic or acquired drug resistance. Our understanding about the mechanisms underlying cisplatin resistance has largely arisen from studies carried out with cancer cell lines in vitro. The process of cisplatin resistance appears to be multifactorial and includes changes in drug transport leading to decreased drug accumulation, increased drug detoxification, changes in DNA repair and damage bypass and/or alterations in the apoptotic cell death pathways. Translation of these preclinical findings to the clinic is emerging, but still scarce. The present review describes and discusses the clinical relevance of in vitro models by comparing the preclinical findings to data obtained in clinical studies.     

Cellular accumulation and DNA interaction studies of cytotoxic <Emphasis Type="Italic">trans</Emphasis>-platinum anticancer compounds

Forty years after the discovery of the anticancer effects of cisplatin, scientists are still pursuing the development of platinum complexes with improved properties regarding side effects and resistance, which are two main problems in cisplatin treatment. Among these compounds, trans-configured platinum complexes with oxime ligands emerged as a new class with features distinct from those of established anticancer agents, including different DNA binding behavior, increased cellular accumulation, and a different pattern of protein interaction. We report herein on the reactivity with biomolecules of three novel pairs of cis- and trans-configured acetone oxime platinum(II) complexes and one pair of 3-pentanone oxime platinum(II) complexes. Cellular accumulation experiments and in vitro DNA platination studies were performed and platinum contents were determined by inductively coupled plasma mass spectrometry. The trans-configured complexes were accumulated in SW480 cells in up to 100 times higher amounts than cisplatin and up to 50 times higher amounts than their cis-configured counterparts; r _b values (number of platinum atoms per nucleotide) were more than tenfold increased in cells treated with trans complexes compared with cells treated with cisplatin. The interaction of the complexes with DNA was studied in cell-free experiments with plasmid DNA (pUC19), in capillary zone electrophoresis with the DNA model 2-deoxyguanosine 5′-monophosphate, and in in vitro experiments showing the degree of DNA damage in the comet assay. Whereas incubation with cis compounds did not induce degradation of DNA, the trans complexes led to pronounced strand cleavage.