MKP1/CL100 controls tumor growth and sensitivity to cisplatin in non-small-cell lung cancer

Non-small-cell lung cancer (NSCLC) represents the most frequent and therapy-refractive sub-class of lung cancer. Improving apoptosis induction in NSCLC represents a logical way forward in treating this tumor. Cisplatin, a commonly used therapeutic agent in NSCLC, induces activation of N-terminal-c-Jun kinase (JNK) that, in turn, mediates induction of apoptosis. In analysing surgical tissue samples of NSCLC, we found that expression of MKP1/CL100, a negative regulator of JNK, showed a strong nuclear staining for tumor cells, whereas, in normal bronchial epithelia, MKP1 was localized in the cytoplasm as well as in nuclei. In the NSCLC-derived cell lines H-460 and H-23, we found that MKP1 was constitutively expressed. Expressing a small-interfering RNA (siRNA) vector for MKP1 in H-460 cells resulted in a more efficient activation by cisplatin of JNK and p38 than in the parental cells, and this correlated with a 10-fold increase in sensitivity to cisplatin. A similar response was also observed in H-460 and H-23 cells when treated with the MKP1 expression inhibitor RO-31-8220. Moreover, expression of a siRNA-MKP2, an MKP1-related phosphatase, had no effect on H-460 cell viability response to cisplatin. Tumors induced by H-460 cells expressing MKP1 siRNA grew slower in nu(-)/nu(-) mice and showed more susceptibility to cisplatin than parental cells, and resulted in an impaired growth of the tumor in mice. On the other hand, overexpression of MKP1 in the H-1299 NSCLC-derived cell line resulted in further resistance to cisplatin. Overall, the results showed that inhibition of MKP1 expression contributes to a slow down in cell growth in mice and an increase of cisplatin-induced cell death in NSCLC. As such, MKP1 can be an attractive target in sensitizing cells to cisplatin to increase the effectiveness of the drug in treating NSCLC.     

Resistance to the tubulin-binding agents in renal cell carcinoma: no mutations in the class I beta-tubulin gene but changes in tubulin isotype protein expression.

PURPOSE: The primary purpose of this study was to determine whether mutations of the class I beta-tubulin gene may be implicated in the inherent resistance to tubulin-binding agents (TBA) in renal cancer, with a small number of samples and cell lines also being examined for class I and III beta-tubulin isotype protein expression. EXPERIMENTAL DESIGN: DNA was extracted from 90 renal tumors and the class I beta-tubulin gene analyzed for mutations. For each sample, eight PCRs were used to cover the complete coding sequence with intronic primers ensuring highly homologous pseudogenes were not coamplified. Additionally, expression levels of class I and III beta-tubulin isotypes in 17 matched normal and malignant renal samples and a panel of renal cell carcinoma cell lines with differing intrinsic resistance to the TBAs was examined by Western blotting. RESULTS: Four polymorphic sequence changes of the class I beta-tubulin gene were identified with no mutations. Class I protein expression levels were higher in tumor tissue versus normal tissue, whereas class III expression showed no consistent change. In renal cancer cell lines, a significant correlation between class III isotype expression and vinblastine sensitivity was observed. CONCLUSIONS: These results do not support a role for mutations in the class I beta-tubulin gene in the intrinsic resistance of renal cancer to TBAs. Class III isotype expression may be implicated in resistance in vitro but in vivo, changes in class I isotype expression in renal cell carcinoma tissue may support a role in resistance to the TBAs and warrants further investigation.     

Emodin enhances cisplatin sensitivity in non-small cell lung cancer through Pgp downregulation

Cisplatin resistance is one of the main causes of chemotherapy failure and tumor progression in non-small cell lung cancer (NSCLC). Emodin has been demonstrated to induce NSCLC cell apoptosis and act as a potential cancer therapeutic agent. However, whether emodin could affect NSCLC cell sensitivity toward cisplatin remains unclear. The present study aimed to determine the effect of emodin and cisplatin combination on the chemosensitivity of NSCLC cells. A549 and H460 cells were treated with different concentrations of cisplatin and/or emodin. Cell Counting Kit-8, fluorescence microscopy, immunofluorescence assays and flow cytometry were used to determine cell proliferation, drug efflux, DNA damage level and cell apoptosis, respectively. P-glycoprotein (Pgp) and multidrug resistance-associated protein 1 (MRP1) expression was detected by western blotting. The results demonstrated that emodin and cisplatin inhibited the proliferation of A549 and H460 cells. Furthermore, emodin inhibited the drug efflux in A549 and H460 cells in a dose-dependent manner. In addition, emodin enhanced cisplatin-induced apoptosis and DNA damage in A549 and H460 cells. Emodin also decreased Pgp expression in A549 and H460 cells in a dose-dependent manner; however, it had no effect on MRP1 expression. Taken together, the results from the present study demonstrated that emodin can increase A549 and H460 cell sensitivity to cisplatin by inhibiting Pgp expression. Emodin may therefore be considered as an effective adjuvant for cisplatin treatment.     

MKP1 repression is required for the chemosensitizing effects of NF-κB and PI3K inhibitors to cisplatin in non-small cell lung cancer

Treatment of non-small cell lung cancer (NSCLC) with cisplatin has a level of antitumor activity still modest. We have shown previously that MKP1/DUSP1 inhibits cisplatin-induced apoptosis in NSCLC cells and is overexpressed in tumors from most patients with stage I–II NSCLC. Here, using different NSCLC cell lines we found that MKP1 and NF-κB are differentially expressed. We studied whether targeting MKP1, NF-κB or both affects cisplatin-induced cell death. MKP1 is expressed in H460 and H727 cells. H727 and H1299 cells showed constitutive phosphorylation of Akt and increased NF-κB activity than did H460 cells. H460-MKP1-siRNA-expressing cells (but not H727-MKP1-siRNA or H1299-MKP1-siRNA cells) exhibit a marked increase in cisplatin response compared with parental cells. Treatment with the PI3K inhibitor LY294002 or the NF-κB inhibitor BAY11-7082 enhanced cisplatin antitumor activity in parental H1299 cells but only weakly affected responses of H727 and H460 cells. MKP1-siRNA expression enhanced the chemosensitization effect of LY294002 and BAY11-7082 on H727 and H460 cells. Additionally, NSCLC cell lines with higher NF-κB-constitutive activation were the most sensitive to PS-341 (Bortezomib), a non-specific NF-κB inhibitor. This finding suggests the proteasome as a suitable strategy in treating NSCLC tumors with high constitutive NF-κB activity. Altogether, these results showed that either an activated PI3K/Akt/NF-κB pathway and/or high MKP1 was linked to reduced sensitivity to cisplatin in NSCLC cells. Inhibition of NF-κB or PI3K potently enhanced cisplatin cytotoxicity in cells with endogenous or genetically induced low MKP1 levels. These findings support the potential improvement in cisplatin responses by co-targeting NF-κB or Akt and MKP1.     

Metuzumab enhanced chemosensitivity and apoptosis in non-small cell lung carcinoma

Targeted therapeutics is used as an alternative treatment of non-small cell lung cancer (NSCLC); however, treatment effect is far from being satisfactory, and therefore identification of new targets is needed. We have previously shown that metuzumab inhibit tumor growth in vivo. The present study was performed to investigate the anti-tumor efficacy of metuzumab combined with gemcitabine and cisplatin (GP), paclitaxel and cisplatin (TP) or navelbine and cisplatin (NP) regimens in multiple NSCLC cell lines. Our results demonstrate that, in comparison to single agent metuzumab or GP treated cells, metuzumab combined with GP display inhibitory effects on tumor growth. Furthermore, we found that metuzumab elevated the sensitivity of cell lines to gemcitabine, which was identified by MTT assay. Flow cytometric analysis showed that metuzumab combined with gemcitabine (GEM) treatment led to an obvious G1 arrest and an elevated apoptosis in A549, NCI-H460 and NCI-H520 cells. Western blot analysis also demonstrated a significantly reduced level of cyclin D1, Bcl-2, and an obviously increase level of Bax and full-length caspase-3 in A549, NCI-H460 and NCI-H520 cells treated with metuzumab/gemcitabine combination in comparison with single agent treated cells. In addition, metuzumab/gemcitabine treated A549, NCI-H460 and NCI-H520 cells also demonstrated a significantly increase in deoxycytidine kinase (dCK) protein level compared with single agent metuzumab or gemcitabine treated cells. Xenograft models also demonstrated that this metuzumab/gemcitabine combination led to upregulation of dCK. Taken together, the mechanisms of metuzumab combined with GP repress tumor growth were that the combined treatment significantly inhibited the tumor cell proliferation, apoptosis and cell cycle in vitro and in vivo and at least partially by induction of dCK expression. Our results suggested that metuzumab could significantly enhance chemosensitivity of human NSCLC cells to gemcitabine. Metuzumab/gemcitabine combination treatment may be a potentially useful therapeutic regimen for NSCLC patients.     

The role of irinotecan combined with Cisplatin or Carboplatin in the treatment of advanced non-small-cell lung cancer

Since the 1980s, cisplatin therapy for advanced non-small-cell lung cancer (NSCLC) has shown improvement in patient outcome with respect to overall survival. In the past decade, several new agents, such as the taxanes (paclitaxel and docetaxel), gemcitabine, vinorelbine, and irinotecan, have also shown promising single-agent efficacy in the treatment of advanced NSCLC. Superior efficacy was observed when these 5 agents were used in combination with cisplatin as compared to cisplatin alone for treatment of patients with NSCLC. The toxicity profiles of these 5 agents were found to be largely nonoverlapping with cisplatin. The results of recent randomized trials with different cisplatin-based chemotherapy regimens have shown that platinum-based therapy is still the mainstay for treatment of NSCLC; however, it appears that a chemotherapy efficacy plateau has been reached. Moreover, it has also been shown that for patients unable to tolerate cisplatin, nonplatinum doublets consisting of gemcitabine with either taxanes or vinorelbine are equivalent in efficacy and can be alternatives for first-line treatment of advanced NSCLC. Thus, the development of new and novel strategies is essential for treatment of NSCLC patients. Ongoing trials with vaccines, signal transduction modulators, antiangiogenic agents, and gene therapy in combination with chemotherapy     

Microtubule disruption induced in vivo by alkylation of beta-tubulin by 1-aryl-3-(2-chloroethyl)ureas, a novel class of soft alkylating agents

We have previously reported that 4-tert-butyl-[3-(2-chloroethyl)ureido] benzene (4-tBCEU), a potent cytotoxic agent, modulates the synthesis of tubulins, suggesting that its cytotoxicity may be mediated through an antimicrotubule mechanism. Indeed, 4-tBCEU and its 4-iso-propyl (4-isopropyl [3-(2-chloroethyl)ureido] benzene) and 4-sec-butyl (4-sec-butyl [3-(2-chloroethyl)ureido] benzene) homologues induced disruption of the cytoskeleton and arrest of the cell cycle in G2 transition and mitosis. To better understand the mechanisms responsible for microtubule disruption by 1-aryl-3-(2-chloroethyl)ureas (CEU), we first examined their cytotoxicity on Chinese hamster ovary cells resistant to vinblastine and colchicine due to the expression of mutated tubulins (CHO-VV 3-2). These cells showed resistance to CEU, e.g., 4-tBCEU having an IC50 of 21.3+/-1.1 microM as compared with an IC50 of 11.6+/-0.7 microM for wild-type cells, suggesting a direct effect of the drugs on tubulins. Western blot analysis confirmed the disruption of microtubules and evidenced the formation of an additional immunoreactive beta-tubulin with an apparent lower molecular weight on SDS polyacrylamide gel. Incubation of MDA-MB-231 cells with [urea-14C]-4-tBCEU revealed the presence of a radioactive protein that coincided with the additional beta-tubulin band, indicating that CEU could covalently bind to the beta-tubulin. The 4-tBCEU-binding site on beta-tubulin was identified by competition of the CEU with colchicine, vinblastine, and iodoacetamide, a specific alkylating agent of sulfhydryl groups of cysteine residues. Colchicine, but not vinblastine, prevented the formation of the additional beta-tubulin band, suggesting that 4-tBCEU alkylates either Cys239 or Cys354 residues near the colchicine-binding site. To determine the cysteine residue alkylated by 4-tBCEU, we incubated the radiolabeled drug with human neuroblastoma cells (SK-N-SH) that overexpress the betaIII-tubulin, an isoform where Cys239 is replaced by a serine residue. The results clearly showed that betaIII-tubulin is not alkylated by [urea-14C]-4-tBCEU, suggesting that cysteine 239 residue is essential for the reactivity of 4-tBCEU with beta-tubulin. Taken together, these findings indicate that the mechanism of cytotoxicity of CEU involves microtubule depolymerization through alkylation of beta-tubulin.     

Is class III β-tubulin a predictive factor in patients receiving tubulin-binding agents?

On the basis of preclinical studies that show overexpression of class III beta-tubulin is associated with resistance to tubulin-binding agents, several investigators have addressed the relation between class III beta-tubulin and outcome in patients treated with such agents. High expression of class III beta-tubulin has been found to be correlated either with low response rates in patients treated with regimens containing taxanes or vinorelbine or with reduced survival in patients with non-small-cell lung cancer, in breast, ovarian, and gastric cancers, and in cancers of unknown primary site. Two studies have shown patients with advanced non-small-cell lung cancer receiving paclitaxel whose tumours expressed high levels of class III beta-tubulin had a lower response to paclitaxel and shorter survival, whereas this variable was not found to be predictive in patients receiving regimens without tubulin-binding agents. Conversely, analysis of samples from patients in the JBR-10 trial, which compared adjuvant chemotherapy to no further therapy in operable non-small-cell lung cancer, showed that chemotherapy seemed to overcome the negative prognostic effect of high levels of expression of class III beta-tubulin and the greatest benefit from cisplatin/vinorelbine was seen in patients with high levels of expression of class III beta-tubulin. Further analyses in operable and advanced non-small-cell lung cancer showed a relation between high expression of class III beta-tubulin and baseline factors such as age under 60 years, adenocarcinoma and large-cell carcinoma histologies, and advanced stage of disease. These results suggest that class III beta-tubulin could be both a prognostic and a predictive factor. Large randomised studies are warranted to determine the prognostic or predictive value of class III beta-tubulin in different settings and tumours.     

Sorafenib is efficacious and tolerated in combination with cytotoxic or cytostatic agents in preclinical models of human non-small cell lung carcinoma

Purpose: Sorafenib tosylate (sorafenib, BAY 43-9006, Nexavar®) is a multi-kinase inhibitor that targets tumor cell proliferation and angiogenesis. These studies evaluated the efficacy and tolerability of combinations of sorafenib plus agents used to treat non-small cell lung cancer (NSCLC) using preclinical models of that disease. Methods: Intravenous (iv) vinorelbine and interperitoneal (ip) cisplatin were administered intermittently (q4d × 3) in combination with sorafenib administered orally (po) once daily for 9 days starting on the same day as the standard agent. In studies with sorafenib and gefitinib, both agents were administered po daily for 10 days starting on the same day. Treatment in all studies was initiated against established sc tumors, and each study was conducted in duplicate. Efficacy was assessed as the delay in tumor growth to a specified size (TGD). Results: Vinorelbine (6.7 mg/kg) and sorafenib (40 mg/kg) produced TGDs of 2.4 and 7.8 days, respectively, in the NCI-H460 NSCLC model. Combination therapy produced a 10.0-day TGD with no increase in toxicity. Combination therapy in the NCI-H23 NSCLC model with the highest evaluated dose levels of sorafenib plus cisplatin was well tolerated and produced TGDs equivalent to those produced by cisplatin alone. Lower dose levels of each agent produced approximately additive TGD’s. Combination therapy in the A549 NSCLC model with sorafenib and gefitinib produced TGDs equivalent to that produced by sorafenib alone with no toxicity. Tumor growth in the MDA-MB-231 mammary tumor model, that contains mutations in signal transduction proteins downstream of the EGF receptor (the target of gefitinib) was also inhibited by sorafenib, but not by gefitinib. Conclusion: Concurrent administration of sorafenib and vinorelbine, cisplatin or gefitinib was at least as efficacious as the individual agents alone and was well tolerated. These results support the inclusion of sorafenib in clinical trials in NSCLC employing combinations of both cytotoxic and cytostatic agents.     

Cisplatin influences acquisition of resistance to molecular‐targeted agents through epithelial–mesenchymal transition‐like changes

Chemotherapy with platinum agents is the standard of care for non‐small‐cell lung cancer (NSCLC); however, novel molecular‐targeted agents like gefitinib have been approved for advanced NSCLCs, including recurrent cases previously treated with platinum‐based chemotherapy. Although these agents show antitumor activity through distinct mechanisms and elicit positive initial responses, tumors invariably develop resistance. Recent studies have revealed mechanisms by which both types of agents induce acquired resistance. However, little is known about whether first‐line treatment with either type of agent affects cancer cell susceptibility and development of resistance against subsequent treatment with the other. Using in vitro drug‐resistant NSCLC cell models, we provide evidence that acquired cisplatin resistance may reduce the sensitivity of cancer cells to subsequent treatment with a molecular‐targeted agent. In addition, first‐line cisplatin treatment influenced the mechanism by which cancer cells developed resistance to subsequent treatment with a molecular‐targeted agent. The influence of cisplatin on acquisition of resistance to a molecular‐targeted agent was associated with epithelial–mesenchymal transition (EMT)‐like alterations such as increased expression of mesenchymal markers, morphological change, and AXL tyrosine kinase‐mediated increased cell motility. Our findings indicate that the influence of platinum‐based chemotherapy on molecular‐targeted therapies and the involvement of EMT and EMT‐related effectors should be considered when developing therapeutic strategies using antitumor agents, especially in the context of sequential therapy.     

In vitro establishment of cis-diammine-dichloroplatinum(II) resistant lung cancer cell line and modulation of apoptotic gene expression as a mechanism of resistant phenotype

After exposure of H460 cells to an increasing concentrations of cis-diammine-dichloroplatinum(II) (cisplatin, CDDP) for 6 months, cisplatin resistant cells were isolated (H460/CIS). The biologic behaviors of H460 and H460/CIS cells were tested using animal experiments. Only the resistant cells developed lung metastases despite cisplatin treatment. The characteristics of H460/CIS cells are as follows, MTT analyses revealed that H460/CIS cells were markedly resistant to cisplatin compared with their parental cells. Also, H460/CIS cells exhibited cross-resistance to DNA damaging agents such as doxorubicin (DXR) and etoposide. Cisplatin treatment dramatically increased p53 expression in parental cells but not in H460/CIS cells which expressed basal levels of p53. Without cisplatin treatment, Bcl-2 and Bax were expressed in H460/CIS cells, but not in parental cell. Our data suggested that p53, Bax and Bcl-2 were up-regulated in H460/CIS cells. These changes could explain some of the mechanisms of cisplatin resistance. Thus, H460/CIS could be useful to investigate the mechanisms of drug resistance to cisplatin including apoptotic gene expressions conferring drug resistance, thereby making progress in the treatment of cisplatin-resistant tumor cells.     

Synergistic effects of new chemopreventive agents and conventional cytotoxic agents against human lung cancer cell lines

Non-small cell lung cancer (NSCLC) cells have constitutively high expression of cytosolic phospholipase A2 (cPLA2) and cyclooxygenase (COX) 2. These NSCLC cells also have increased prostaglandin expression (PGE2). Many lung cancers also express 12-lipoxygenase RNA and 12-lipoxygenase protein and biosynthesize 12(S)-hydroxyeicosatetraenoic acid, which correlates with their metastatic potential. Several studies have demonstrated that COX-1 and COX-2 inhibitors could inhibit the in vitro growth of human lung cancer cell lines. In this report, we evaluated the growth-inhibitory effects of sulindac sulfide, a COX-1 and COX-2 inhibitor; exisulind (sulindac sulfone), a novel proapoptotic agent that does not inhibit COX enzymes; and nordihydroguaiaretic acid (NDGA), a lipoxygenase inhibitor on human lung cancer cell lines. We compared these effects with those of 13-cis-retinoic acid, a chemoprevention agent, and with the cytotoxic chemotherapeutic agents paclitaxel and cisplatin, alone or in combination. Our goal was to develop new chemoprevention and treatment strategies. Each of the six agents tested inhibited the in vitro growth of three NSCLC and three SCLC cell lines at the highest concentration. Paclitaxel was the most potent agent (IC50 = 0.003-0.150 microM); sulindac sulfide, NDGA, and 13-cis-retinoic acid had intermediate potency (IC50 = 4-80 microM), and cisplatin and exisulind were the least potent (IC50 = 150-500 microM). Combination studies showed synergistic interactions for sulindac sulfide, exisulind, and NDGA with paclitaxel, cisplatin, and 13-cis-retinoic acid, regardless of drug-resistance phenotype. At high concentrations, the combination of 13-cis-retinoic acid and each of the five other drugs resulted in a strong synergistic effect. These studies provide a rationale for chemoprevention (exisulind +/- retinoic acid +/- NDGA) and therapeutic (exisulind +/- paclitaxel +/- cisplatin) studies in patients at risk for, or with, lung cancer.     

Cisplatin-controlled p53 gene therapy for human non-small cell lung cancer xenografts in athymic nude mice via the CArG elements

Cisplatin, a commonly used chemotherapeutic agent, causes tumor cell death by producing DNA damage and generating reactive oxygen intermediates, which have been reported to activate the early growth response-1 ( Egr-1 ) promoter through specific cis -acting sequences, termed CArG elements. The aim of this study was to construct an adenoviral vector containing CArG elements cloned upstream of the cDNA for human wt-p53 , and to observe the effect of this vector on human non-small cell lung cancer (NSCLC) xenografts in athymic nude mice when combined with cisplatin treatment. The adenoviral vector AdEgr–p53 was generated by inserting CArG elements upstream of human wt-p53 cDNA. Two human NSCLC cell lines of varying p53 gene status, A549 (containing wild-type p53 ) and H358 (containing an internal homozygous deletion of the p53 gene) were used for in vitro and in vivo experiments. Wt-p53 production in cultured tumor cells and xenografts treated with the combination of AdEgr–p53 and cisplatin were detected by enzyme-linked immunosorbent assays. The antitumor responses in nude mice with the A549 or H358 xenografts following treatment with AdEgr–p53 and cisplatin were observed. We found that p53 was produced in tumor cells and xenografts treated with a combination of AdEgr–p53 and cisplatin. Furthermore, the Egr-1 promoter is induced by cisplatin, and this induction is mediated in part through the CArG elements. There was an enhanced antitumor response without an increase in toxicity following treatment with AdEgr–p53 and cisplatin, compared with either agent alone. Cisplatin-inducible p53 gene therapy may provide a means to control transgene expression while enhancing the effectiveness of commonly used chemotherapeutic agents. This is a novel treatment for human NSCLC. ( Cancer Sci 2005; 96: 706 – 712)     

Mitotic arrest deficient 2 expression induces chemosensitization to a DNA-damaging agent, cisplatin, in nasopharyngeal carcinoma cells

Recently, mitotic arrest deficient 2 (MAD2)-mediated spindle checkpoint is shown to induce mitotic arrest in response to DNA damage, indicating overlapping roles of the spindle checkpoint and DNA damage checkpoint. In this study, we investigated if MAD2 played a part in cellular sensitivity to DNA-damaging agents, especially cisplatin, and whether it was regulated through mitotic checkpoint. Using nine nasopharyngeal carcinoma (NPC) cell lines, we found that decreased MAD2 expression was correlated with cellular resistance to cisplatin compared with the cell lines with high levels of MAD2. Exogenous MAD2 expression in NPC cells also conferred sensitivity to DNA-damaging agents especially cisplatin but not other anticancer drugs with different mechanisms of action. The increased cisplatin sensitivity in MAD2 transfectants was associated with mitotic arrest and activation of apoptosis pathway evidenced by the increased mitotic index and apoptosis rate as well as decreased Bcl-2 and Bax ratio and expression of cleaved poly(ADP-ribose) polymerase and caspase 3. Our results indicate that the MAD2-induced chemosensitization to cisplatin in NPC cells is mediated through the induction of mitotic arrest, which in turn activates the apoptosis pathway. Our evidence further confirms the previous hypothesis that spindle checkpoint plays an important part in DNA damage-induced cell cycle arrest and suggests a novel role of MAD2 in cellular sensitivity to cisplatin.     

Clinical relevance of the homologous recombination machinery in cancer therapy

Cancer chemotherapy and radiotherapy kill cancer cells by inducing DNA damage, unless the lesions are repaired by intrinsic repair pathways. DNA double-strand breaks (DSB) are the most deleterious type of damage caused by cancer therapy. Homologous recombination (HR) is one of the major repair pathways for DSB and is thus a potential target of cancer therapy. Cells with a defect in HR have been shown to be sensitive to a variety of DNA-damaging agents, particularly interstrand crosslink (ICL)-inducing agents such as mitomycin C and cisplatin. These findings have recently been applied to clinical studies of cancer therapy. ERCC1, a structure-specific endonuclease involved in nucleotide excision repair (NER) and HR, confers resistance to cisplatin. Patients with ERCC1-negative non-small-cell lung cancer were shown to benefit from adjuvant cisplatin-based chemotherapy. Imatinib, an inhibitor of the c-Abl kinase, has been investigated as a sensitizer in DNA-damaging therapy, because c-Abl activates Rad51, which plays a key role in HR. Furthermore, proteins involved in HR have been shown to repair DNA damage induced by a variety of other chemotherapeutic agents, including camptothecin and gemcitabine. These findings highlight the importance of HR machinery in cancer therapy. ( Cancer Sci 2008; 99: 187–194)     

Heat shock protein 72 does not modulate ionizing radiation-induced apoptosis in U1810 non-small cell lung carcinoma cells

Heat shock proteins (HSP) have been shown to interfere with apoptosis signaling, suggesting that there might be a role for these proteins as mediators of resistance to ionizing radiation (IR)-induced apoptosis. Protein expression of the stress inducible heat shock proteins, HSP72 and HSP27, was analyzed in a panel of lung carcinoma cell lines displaying various degrees of radiosensitivity. Expression of HSP72 was high in all cell lines investigated while HSP27 was present in all non-small cell lung carcinoma (NSCLC) and 6/9 small cell lung carcinoma (SCLC) cell lines. Heat shock, but not IR, induced or further increased the expression of HSP27 and HSP72. Moreover, elevation of heat shock protein level prior to irradiation did not attenuate IR-induced apoptotic signaling or the induction of apoptosis. Protein level of HSP72 was downregulated in a radioresistant NSCLC cell line by RNA interference. However, this did not sensitize cells to treatment with DNA-damaging agents such as IR, cisplatin or VP16. Thus, the results from this first study on the relationship between stress-inducible HSP expression and IR-induced apoptosis in lung cancer cells do not support a role for HSP 27 and 72 in the radioresistance of NSCLC cells.     

Association of p53 gene mutations with sensitivity to TZT-1027 in patients with clinical lung and renal carcinoma

BACKGROUND: It has been revealed that chemotherapy using DNA-damaging agents and radiotherapy were influenced by the p53 status of tumors; however, p53 status did not influence chemotherapy using antimicrotubule agents. To elucidate whether a novel antimicrotubule agent, TZT-1027, is influenced by the p53 status of tumors, the authors investigated the sensitivities of specimens obtained from patients with nonsmall cell lung carcinoma (NSCLC) and renal cell carcinoma (RCC) to various anticancer agents, including TZT-1027, and the status of the p53 gene in those specimens. METHODS: Twenty-nine NSCLC specimens and 22 RCC specimens were analyzed for their sensitivity to various anticancer agents and their p53 status. Sensitivities of the specimens to nine anticancer agents were determined by flow cytometric analysis. To determine p53 status, polymerase chain reaction amplification with primers for exons 5--9 was conducted, and the products were subjected to single-strand conformation polymorphism analysis. RESULTS: In the NSCLC specimens, anticancer agents, including TZT-1027, showed strong antitumor activity against 50--75% of specimens with the wild type p53 gene. TZT-1027 showed strong antitumor activity against 40% of specimens with the mutant type p53 gene, whereas DNA-damaging agents showed such activity only in 16--28% of specimens. In RCC specimens, TZT-1027 showed potent antitumor activity in 29% of specimens with the wild type gene, and DNA-damaging agents showed such activity in 6--18% of specimens. TZT-1027 showed strong antitumor activity in 40% of specimens with the mutant type p53 gene, whereas DNA-damaging agents showed such activity only in 0--20% of specimens. CONCLUSIONS: We found evidence to suggest that TZT-1027 was influenced less by the p53 status of specimens than DNA-damaging agents. Therefore, TZT-1027 is expected to show similar antitumor activity against tumors with a loss of p53 function as well as those with normal function of p53 in clinical fields.     

慢病毒介导β4整合素shRNA对人非小细胞肺癌H460SM细胞皮下移植瘤生长的影响

目的:研究慢病毒介导的β4整合素(integrinβ4,ITGB4) shRNA对人非小细胞肺癌(non-small cell lung cancer,NSCLC)裸鼠皮下移植瘤生长的抑制作用.方法:采用实时荧光定量-PCR (real-time fluorogenic quantitative-PCR,RFQ-PCR)和蛋白质印迹法检测ITGB4在不同人NSCLC细胞中的表达水平;慢病毒介导ITGB4 shRNA抑制NSCLC H460SM细胞中ITGB4的表达,再用RFQ-PCR和蛋白质印迹法检测ITGB4的表达水平,评价基因沉默效率;裸鼠随机分为3组,皮下分别接种H460SM-NS细胞(对照组)、稳定表达ITGB4 shRNA的H460SM-68和H460SM-71细胞.每隔1d测量裸鼠体质量和肿瘤大小,比较各组裸鼠肿瘤生长情况.处死裸鼠,剥离肿瘤,测量肿瘤大小和质量.结果:ITGB4在大多数人NSCLC细胞中均有表达,其中大细胞肺癌H460SM和NCI-H460细胞中ITGB4表达量明显高于NCI-H661细胞(P＜0.01),H460SM细胞中ITGB4表达水平明显高于亲本NCI-H460细胞(P＜0.01):ITGB4 shRNA转染组细胞中ITGB4的表达水平明显低于阴性对照组细胞(P＜0.01); ITGB4 shRNA转染组皮下移植瘤生长速度明显低于阴性对照组(P＜0.01).结论:ITGB4表达可能与人NSCLC细胞的致瘤、侵袭、转移的表型有关;抑制ITGB4的表达,可以抑制人NSCLC细胞H460SM裸鼠皮下移植瘤的生长.     

ATR mediates cisplatin resistance in a p53 genotype-specific manner

The protein kinase encoded by the ataxia telangiectasia and Rad3-related (ATR) gene is activated by DNA-damaging agents that are frequently used as anticancer therapeutics. Inhibition of ATR expression in cultured cancer cells has been demonstrated to increase sensitivity to chemotherapeutic drugs, including the DNA-crosslinking agent cisplatin. Cisplatin is a widely used and effective drug, but its use is associated with significant toxicity. Here, we demonstrate that genetic inhibition of ATR expression selectively enhanced cisplatin sensitivity in human colorectal cancer cells with inactivated p53. A knock-in strategy was used to restore wild-type p53 in cells harboring wild-type or mutant ATR alleles. Knock-in of functional p53 in ATR-deficient cells restored checkpoint function, suppressed apoptotic pathways and markedly increased clonogenic survival after cisplatin treatment. These results suggest that a strategy that combines specific inhibitors of ATR and conventional therapies might promote synthetic lethality in p53-deficient tumors, and thus minimize toxicity to normal tissues.     

不同铂类联合紫杉类或长春瑞滨对非小细胞肺癌荷瘤裸鼠抑瘤作用的实验研究*

目的　探讨洛铂（ＬＢＰ）、顺铂（ＤＤＰ）和卡铂（Ｃａｂ）单用或联合紫杉醇（ＰＴＸ）、多西他赛（ＤＯＣ）和长春瑞滨（ＮＶＢ）对非小细胞肺癌（ＮＳＣＬＣ）荷瘤裸鼠的抑瘤作用。方法　选用人大细胞肺癌细胞株ＮＣＩ-Ｈ４６０接种于裸小鼠皮下成瘤。单药抑瘤实验中设ＬＢＰ（３.７５、７.５、１５ｍｇ／ｋｇ）３组及Ｃａｂ（６０ｍｇ／ｋｇ）、ＤＤＰ（５ｍｇ／ｋｇ）各１组;联合抑瘤实验设ＬＢＰ（７.５ｍｇ／ｋｇ）、ＤＰＰ（２.５ｍｇ／ｋｇ）分别与ＤＯＣ（５ｍｇ／ｋｇ）、ＰＴＸ（１２ｍｇ／ｋｇ）、ＮＶＢ（５ｍｇ／ｋｇ）联合共６组以及各自单药５组,每组均随机分配６只成瘤小鼠,经静脉ｄ０、ｄ７给药（ＰＴＸｄ０、ｄ２、ｄ４给药除外）。各设１组对照组,每组１２只。每周２～３次记录瘤体体积和裸鼠体重,并计算相对肿瘤增殖率（Ｔ／Ｃ）。结果　（１）单药抑瘤实验中,ＬＢＰ抑瘤效果与剂量相关,中、高剂量组的Ｔ／Ｃ分别为５１.１％和３６.３％,低于Ｃａｂ组（５１.６％）,但ＬＢＰ高剂量组有１只小鼠出现药物相关性死亡。对裸鼠体重的影响由大到小依次为：ＤＤＰ组＞ＬＢＰ高剂量组＞ＬＢＰ中剂量组＞Ｃａｂ组＞ＬＢＰ低剂量组。（２）联合抑瘤实验中,ＬＢＰ＋ＰＴＸ组的Ｔ／Ｃ最低,为２４.4％,明显优于ＤＤＰ＋ＰＴＸ组（Ｐ＜０.０５）;ＬＢＰ＋ＤＯＣ组和ＤＤＰ＋ＤＯＣ组亦对小鼠体重有明显影响。结论　ＬＢＰ单药对ＮＣＩ Ｈ４６０荷瘤小鼠的抑瘤作用呈剂量依赖性,介于ＤＤＰ与Ｃａｂ之间;而ＬＢＰ联合ＰＴＸ的抑瘤作用最强,明显优于ＤＤＰ联合ＰＴＸ及其他两药联合。