Inactivation of p53 Sensitizes Astrocytic Glioma Cells to BCNU and Temozolomide,but not Cisplatin

Summary p53 inactivation sensitizes U87MG astrocytic glioma cells to 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and temozolomide (TMZ), drugs used clinically to treat high-grade astrocytomas. In this report, we examined the effect of p53 inactivation on the chemosensitivity of two additional human astrocytic glioma cell lines, D54 and A172, in order to assess whether sensitization is a general property of astrocytic tumor cells. Compared to control cells with intact p53 function, derived lines in which p53 was inactivated displayed significantly reduced clonogenic survival after exposure to BCNU and TMZ. Sensitization to both BCNU and TMZ was associated with failure of p21^WAF1 induction, lack of a sustained G2 cell cycle arrest and significant tumor cell death. These findings suggest that enhanced sensitivity to BCNU and TMZ is a general property of human astrocytic glioma cells in which p53 was disrupted. In contrast, p53 inactivation rendered D54 and U87MG cells significantly more resistant to cis-dichlorodiamminoplatinum (CDDP), another chemotherapeutic to which high-grade astrocytomas sometimes respond. These results indicate that p53 status influences the chemosensitivity of astrocytic glioma cells in a drug-type specific manner, a finding that may have implications for the selection of drug treatments for patients with astrocytic gliomas.     

Combination therapy of 2-5A antisense against telomerase RNA and cisplatin for malignant gliomas

2',5'-Oligoadenylate (2-5A) linked to an antisense oligonucleotide against human telomerase RNA (2-5A-anti-hTR) is a novel therapeutic modality we have recently developed. We designed the oligonucleotide to target telomerase which maintains chromosome integrity in cancer cells. We have already demonstrated the efficacy of this new therapy in malignant glioma cells with telomerase activity. In the present study, we investigated the effect of 2-5A-anti-hTR in combination with cisplatin, an anti-cancer drug commonly used for malignant glioma patients. Six human malignant glioma cell lines with telomerase activity were treated with 0.5 microM 2-5A-anti-hTR and/or cisplatin (0, 0.1, 1, 5, 10, or 20 microg/ml) for three days, and cell viability was measured using the MTT colorimetric assay. The combination therapy showed synergistic effect at 1 microg/ml cisplatin and additive effect at 5 microg/ml cisplatin. TUNEL staining of the treated cells showed significantly increased apoptotic cells after the combination therapy. Furthermore, tumor growth of subcutaneous xenografts of human malignant glioma cells in nude mice was effectively reduced after the combination therapy for seven days. Our study shows that the tumor-killing effects of the combined treatments are, at least in part, due to induction of apoptosis. 2-5A-anti-hTR is a promising therapy not only when used alone, but also in combination with cisplatin.     

Thresholds of O6-alkylguanine-DNA alkyltransferase which confer significant resistance of human glial tumor xenografts to treatment with 1,3-bis(2-chloroethyl)-1-nitrosourea or temozolomide.

Bis-2-chloroethylnitrosourea (BCNU) or temozolomide (TMZ) were tested alone or in combination with the AGT inhibitors O6-benzyl-2'-deoxyguanosine (dBG) or O6-benzylguanine (BG) against human glial tumor xenografts growing s.c. in athymic mice. Four glioblastoma (SWB77, SWB40, SWB39, and D-54) and one anaplastic oligodendroglioma (SWB61) xenografts having O6-alkylguanine-DNA alkyltransferase (AGT) activities of 75, 45, 10, < 10, and 16 fmol/mg protein, respectively, were used. BCNU at 35 mg/m2 was ineffective against these tumors, although 70 mg/m2 (LD10, 75 mg/m2) produced a marked tumor growth delay (T-C) in D54 but had no effect against SWB40 or SWB77. Coadministration of BG or dBG and BCNU necessitated reduction of the BCNU dose to a maximum of 30 and 35 mg/m2, respectively, because of increased toxicity. Optimized treatment with dBG (250 mg/m2) and BCNU (35 mg/m2) resulted in T-Cs of 30, 29, 11, 16, and 14 days for SWB77, SWB40, SWB39, D-54 and SWB61, respectively. These delays were more pronounced than those induced with optimized, isotoxic treatments with BG (180 mg/m2) and BCNU (30 mg/m2). In comparison to BCNU, TMZ was less toxic, with an LD10 of 400 mg/m2. TMZ (300 mg/m2) was more effective than BCNU against SWB77, SWB40, and SWB61, inducing T-Cs of 23, 53, and 56 days, respectively. BG and dBG enhanced the toxicity of TMZ in athymic mice by decreasing the LD10 from 400 to 200 mg/m2. TMZ (180 mg/m2) with either BG (180 mg/m2) or dBG (250 mg/m2) resulted in T-Cs of 31 and 49 days in SWB77, respectively, as compared with 16 days for TMZ (180 mg/m2) alone. In SWB40, the combination of TMZ with dBG, but not with BG, was significantly more effective than the maximum tolerated dose of TMZ (300 mg/m2) alone. The combination of TMZ with AGT inactivators had no benefit, as compared with TMZ alone, against xenografts with marginal AGT activity. In conclusion, at equimolar doses dBG was less toxic than BG in athymic mice when combined with either BCNU or TMZ. In this regard, BCNU or TMZ can be used at higher doses in combination with dBG than with BG. This study further demonstrates that there is a significant benefit of depleting AGT with nonspecific AGT inhibitors prior to treatment with either BCNU or TMZ in tumors having AGT activity >45 fmol/mg protein.     

Treatment of prostate cancer in vitro and in vivo with 2-5A-anti-telomerase RNA component

Prostate cancer is the most common malignancy of elderly men in the United States. Since there is no curative treatment for advanced prostate cancer, exploration of novel modalities of treatment is essential. Telomerase, a ribonucleoprotein, is detected in the vast majority of prostate cancer, but not in normal or benign prostatic hyperplasia tissues. Thus, telomerase is expected to be a very strong candidate for targeted therapy of prostate cancer. In this study, we synthesized a 19-mer antisense oligonucleotide against the RNA component of human telomerase (hTR) linked to a 2-5A molecule (2-5A-anti-hTR) and examined its cytotoxic effect on prostate cancer cells. The 2-5A antisense strategy relies on the recruitment and activation of RNase L at the site of targeted RNA sequence. We here show that treatment with 2-5A-anti-hTR in the presence of a cationic liposome reduced cell viability of tumor cell lines tested to 9-18% within 6 days. In contrast, normal fibroblast cells were resistant to the treatment. Its effect was mainly due to induction of apoptosis by activated caspase family members. Furthermore, treatment of subcutaneous tumors in nude mice with 2-5A-anti-hTR significantly suppressed the tumor growth through induction of apoptosis (P<0.001). The treatment with 2-5A-anti-hTR may be a promising strategy for the treatment modality of prostate cancer with telomerase activity.     

Effect of fluosol-DA/carbogen on etoposide/alkylating agent antitumor activity

Summary The tumor growth delay produced by the combination of etoposide with the alkylating agent CDDP or BCNU and Fluosol-DA with carbogen breathing in three model tumor systems was examined. The addition of Fluosol-DA to etoposide treatment increased tumor growth delay 2.8-fold, 3.3-fold and 2.2-fold in the FSaIIC fibrosarcoma, the Lewis lung carcinoma and the SW2 small-cell xenograft, respectively. In both the FSaIIC fibrosarcoma and the Lewis lung carcinoma the combination of etoposide treatment with CDDP produced an additive effect. When Fluosol-DA was added to this combination the tumor growth delay increased 1.9-fold and 1.4-fold in the FSaIIC fibrosarcoma and the Lewis lung carcinoma, respectively. Adding Fluosol-DA to a treatment regimen with etoposide and BCNU produced a 2.2-fold, 2.0-fold and 1.6-fold increase in the tumor growth delay of the FSaIIC fibrosarcoma, the Lewis lung carcinoma and the SW2 small-cell xenograft, respectively. The effect of these various treatment combinations on tumor cell survival was assessed in the FSaIIC fibrosarcoma. When the alkylating agents CDDP or BCNU were prepared in Fluosol-DA, there was an additional increase in tumor cell kill, so that with CDDP there was 2.1-fold and 4.7-fold increase in tumor cell kill and with BCNU there was 1.5-fold and 1.2-fold increase in tumor cell kill compared to the drug plus Fluosol-DA and the drug plus Fluosol-DA/carbogen breathing, respectively. The combination of etoposide and CDDP led to less than additive cell killing, and the combination of etoposide and BCNU appeared to be additive, as predicted by simple product summation, in all of the treatment conditions examined. Both etoposide + CDDP and etoposide + BCNU produced additive or less than additive toxicity to bone marrow as measured by CFU-GM.This work was supported by NCI grant # 2PQ1-CA19589-10 (BAT), RO1-CA36498-03 (SDB), grants from the American Cancer Society of New York (CH 340) and Alpha Therapeutics Co., Los Angeles,CA 90032 (BAT) and NIH fellowship # 5F32-CA07821-02 (SAH)     

A Phase I and pharmacokinetic study of temozolomide and cisplatin in patients with advanced solid malignancies.

Temozolomide (TMZ) is an oral imidazotetrazinone that is spontaneously converted to 5-(3-methyltriazen-1-yl)imidazole-4-carboxamide (MTIC) at physiological pH. MTIC methylates DNA at the O6 position of guanine, although this lesion may be repaired by the enzyme O6-alkylguanine-DNA alkyltransferase (AGAT). In this study, TMZ was combined with cisplatin (CDDP), because both agents have single-agent activity against melanoma and other tumor types. Additionally, CDDP has been shown to inactivate AGAT, and subtherapeutic concentrations of CDDP have been shown to increase the sensitivity of leukemic blasts to TMZ. This Phase I study sought to determine the toxicities, recommended dose, and pharmacological profile of the TMZ/CDDP combination. Patients were treated with oral TMZ daily for 5 consecutive days together with CDDP on day 1 (4 h after TMZ) every 4 weeks at the following TMZ (mg/m2/day)/CDDP (mg/m2) dose levels: 100/75, 150/75, 200/75, and 200/100. Plasma samples were obtained on days 1 and 2 to evaluate the pharmacokinetic parameters of TMZ alone and in combination with CDDP. Fifteen patients received a total of 44 courses of TMZ/CDDP. The principal toxicities of the regimen consisted of neutropenia, thrombocytopenia, nausea, and vomiting, which were intolerable in two of six new patients treated at the 200/100 mg/m2 dose level. Of five patients receiving 17 courses at the next lower dose level (200/75 mg/m2), none experienced dose-limiting toxicity. Antitumor activity was observed in patients with non-small cell lung cancer, squamous cell carcinoma of the tongue, and leiomyosarcoma of the uterus. Pharmacokinetic studies of TMZ revealed the following pertinent parameters (mean +/- SD): time to maximum plasma concentration (Tmax) = 1.1+/-0.6 h (day 1) and 1.7+/-0.9 h (day 2); elimination half-life (t1/2) = 1.74+/-0.22 h (day 1) and 2.35+/-0.70 h (day 2); and clearance (Cl(s)/F) = 115+/-27 ml/min/m2 (day 1) and 141+/-109 ml/min/m2 (day 2). TMZ drug exposure, described by the area under the plasma concentration-time curve (AUCinfinity) and the maximum plasma concentration (Cmax), was similar on days 1 and 2. On the basis of these results, the recommended doses for Phase II clinical trials are TMZ 200 mg/m2/day for 5 days with 75 mg/m2 CDDP on day 1, every 4 weeks. The addition of CDDP did not affect the tolerable dose of single-agent TMZ (200 mg/m2/day x 5 days), nor did it substantially alter the pharmacokinetic behavior of TMZ.     

Mechanisms operative in the antitumor activity of temozolomide in glioblastoma multiforme

PURPOSE: Glioblastoma multiforme (GBM) is the most frequent and incurable brain tumor in adults. Although temozolomide (TMZ) does not cure GBM, it has demonstrated anti-GBM activity and has improved survival (8-14 months) and quality of life. We studied the mechanisms by which TMZ affects 2 human GBM cell lines; U251-MG and U87-MG, aiming to unravel the drug-activated cascades to enable the development of combination therapies that will improve the efficacy of TMZ. MATERIALS AND METHODS: The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium assay was used to assess cell viability. Modulation of gene expression by TMZ therapy was assayed by gene profiling and verified by quantitative real-time polymerase chain reaction. Protein levels influenced by the treatment were studied by Western blots and immunocytochemistry. RESULTS: Increasing concentrations of TMZ decreased cell viability in a concentration-dependent manner. The expression of 1,886 genes was altered >2-fold after TMZ treatment. We focused on the 81 genes similarly altered by TMZ treatment in both cell lines to neutralize tissue-specific characteristics. Fourteen target genes of hypoxia-inducible factor (HIF-1), were found to be up-regulated after TMZ treatment including vascular endothelial growth factor (VEGF). HIF-1alpha expression was constant at the mRNA level; however, its post-treatment protein levels increased compared with those of untreated control cells. DISCUSSION: The genetic analyses suggest that treatment with TMZ activates stress mechanisms in GBM cells that include the angiogenesis-inducing proteins HIF-1alpha and VEGF. We propose that treatment with TMZ be supplemented with either an antibody to VEGF or down-regulators of HIF-1alpha to improve clinical results of TMZ in the treatment of GBM.     

Phase II Trial of Cisplatin Plus Temozolomide, in Recurrent and Progressive Malignant Glioma Patients

We report a phase II trial of cisplatinum and temozolomide (TMZ) combination in recurrent malignant glioma patients. The DNA repair protein O(6)-alkylguanine-DNA alkyltransferase (AGAT) is important in glioblastoma resistance to alkylating antitumor agents. In vitro, cisplatin (CDDP) decreases MGMT activity in a time- and dose-dependent manner. Thirty-three recurrent malignant glioma patients (20 GBM-13 AA) were treated at recurrence or progression with a CDDP and TMZ association. On days 1 and 2, iv CDDP (40 mg/sqm) was administered. TMZ (at the dose of 200 mg/sqm) was administered as a single oral daily-dose on days 2-6 (starting 24 h after the first CDDP dose), the cycle was repeated every 4 weeks. All patients had been previously treated with surgery followed by radiotherapy and CDDP + BCNU chemotherapy. The primary endpoint of the study was progression free survival at 6 months (PFS-6). Secondary endpoints included radiological response and toxicities. Thirty-three patients received a total of 113 courses (median 3 range 1-11). Complete responses were not observed, partial responses were 18.8% with an additional 39.9% of stable disease. For the whole group of patients the PFS at 6 and 12 months was 52% and 15% with a median TTP of 33 weeks. PFS-6 for GBM and Anaplastic astrocytoma (AA) were 35% and 69%, respectively. PFS-12 for GBM and AA were 13.8% and 17.3%, respectively. Median TTP was 21.3 and 39.5 weeks, respectively. The principal toxic effects of the regimen were: neutropenia (5 WHO grade IV), thrombocytopenia (4 WHO grade IV), nausea and vomiting.     

Temozolomide and cisplatin versus temozolomide in patients with advanced melanoma: a randomized phase II study of the Hellenic Cooperative Oncology Group.

PURPOSE: Temozolomide (TMZ) is an oral alkylating agent that produces methyl adducts at the 0.6 position of guanine. The methyl adducts are removed by the DNA repair enzyme AGAT. As demonstrated by in vitro studies, cisplatin (CDDP) is able to down-regulate the AGAT activity, suggesting that CDDP could enhance the antitumor activity of TMZ. We designed a randomized phase II study to evaluate and compare the activity and safety profile of the combination versus single-agent TMZ in patients with advanced melanoma. PATIENTS AND METHODS: From January 2000 to April 2002, 132 patients were enrolled on the study. Patient and tumor characteristics were well balanced between the two arms. Patients with cerebral metastases were included. Patients received TMZ 200 mg/m(2)/day orally for five consecutive days every 4 weeks or TMZ + CDDP 200 mg/m(2) daily on days 1-5 and 75 mg/m(2) of CDDP on day 1. RESULTS: Tumor responses (complete and partial responses) were seen in 16 patients (26%) in arm A and 19 patients (29%) in arm B. The median time to progression (TTP) was 3.8 months in arm A and 5.8 months in arm B. The median overall survival (OS) was 11.5 months in arm A and 12 months in arm B. The difference between treatment arms regarding objective response rates, TTP and OS were not statistically significant. Toxicity was comparable between the two arms for anemia, leukopenia, neutropenia, thrombocytopenia, fatigue, constipation and arthralgias/myalgias. There was significantly more grade 3 and 4 emesis in the combination arm. CONCLUSIONS: No clear benefit in terms of response rates, median TTP or OS was shown with the combination of TMZ + CDDP. Additionally, the combination was associated with higher incidence of grade 3 and 4 emesis.     

Synergy of enediyne antibiotic lidamycin and temozolomide in suppressing glioma growth with potentiated apoptosis induction

The present work evaluated the synergistic efficacy of an enediyne antibiotic lidamycin (LDM) plus temozolomide (TMZ) against glioma in vitro and in vivo. LDM plus TMZ inhibited the proliferations of rat glioma C6 cells and human glioma U87 cells more efficiently than the single usage of LDM or TMZ. In addition, LDM also potentiated the apoptosis inductions by TMZ in rat C6 cells and human U87 cells. Meanwhile, the results of TdT-mediated dUTP Nick End Labeling assay for subcutaneous U87 tumor sections indicated an enhanced apoptosis induction in vivo by LDM plus TMZ, which confirmed the high potency of the combination for glioma therapy. As determined by Western blot, apoptosis signal pathways in C6 cells and U87 cells were markedly affected by the synergistic alteration of P53, bax, procaspase 3, and bcd-2 expression. In both subcutaneous U87 xenograft and C6 intracerebral orthotopic implant model, TMZ-induced glioma growth suppression was dramatically potentiated by LDM. As shown, the combination therapy efficiently reduced the tumor volumes and tumor weights of the human glioma U87 xenograft. Kaplan–Meier assay revealed that LDM plus TMZ dramatically prolonged the life span of C6 intracerebral tumor-bearing rats with decreased tumor size. This study indicates that the combination of LDM with TMZ might be a promising strategy for glioma therapy.     

不同化疗药物对胶质瘤获得性SLC22A18基因耐药的逆转作用

目的:探讨临床上治疗胶质瘤的不同化疗药物对胶质瘤U251细胞获得性SLC22A18耐药的逆转作用及可能的分子机制。方法:将重组腺病毒载体(Ad)介导的SLC22A18基因联合替莫唑胺(TMZ)、卡氮芥(BCNU)以及顺铂(DDP)3种常见化疗药物处理对Ad/SLC22A18产生耐药的U251-SLC22A18/R胶质瘤细胞,通过MTT比色法检测处理后胶质瘤细胞的存活率,以评估体外不同化疗药物对SLC22A18耐药的逆转作用;在体内进一步评估该逆转策略的有效性;并且通过免疫印迹等方法探讨逆转耐药的可能的分子机制。结果:在体外只有TMZ和BCNU能够使U251-SLC22A18/R细胞对Ad/SLC22A18重新敏感。进一步的研究结果表明联合TMZ和Ad/SLC22A18能在体内有效地抑制U251-SLC22A18/R细胞来源的胶质瘤生长,且联合TMZ和Ad/SLC22A18抑制作用明显比其它对照组强。Ad/SLC22A18和TMZ的联合治疗可以下调MGMT蛋白的表达,并且Ad/SLC22A18和BCNU的联合治疗可以上调Bax蛋白的表达。结论:联合应用Ad/SLC22A18和TMZ或BCNU能在体内外有效地逆转U251-SLC22A18/R细胞对SLC22A18的获得性耐药,TMZ的逆转作用可能与其诱导的MGMT蛋白低表达有关,BCNU的逆转作用可能与其诱导的Bax蛋白过度表达有关。     

Synergistic inhibitory effect of wogonin and low-dose paclitaxel on gastric cancer cells and tumor xenografts

Objective：To investigate the synergistic inhibitory effects of wogonin （WOG） and chemotherapeutic drugs on growth of gastric cancer cells and tumor xenografts.Methods：The IC50 values of WOG,cisplatin （CDDP） and paclitaxel （PTX） in four gastric cancer cell lines were determined by MTS assay.Hoechst staining and the median effect method of Chou-Talalay were used to assess the apoptosis of cells and the interaction of two drugs,respectively.BGC-823-derived xenografts in nude mice were established to investigate the effects of WOG combined with chemotherapeutic drugs in vivo.Results：WOG,CDDP and PTX inhibited the growth of BGC-823,MGC-803,MKN-45 and HGC-27 gastric cancer cells in a dose-dependent manner.WOG combined with CDDP or PTX synergistically inhibited the growth of all gastric cancer cell lines in vitro.In BGC-823,MGC-803,HGC-27 and MKN-45 cell lines,synergisms between WOG and PTX were shown when the fraction affected （Fa） values were ＜0.45,＜0.90,＜0.85 and ＜0.60.While WOG and CDDP had a synergistic inhibitory.effect when the Fa values were ＞0,＞0,＞0.65 and ＞0.10.From the results of in vivo experiments using tumor xenografts,WOG and low-dose PTX showed better efficacy than either drug alone.The inhibitory percentages of tumor weight were 61.58％,20.29％,and 22.28％ for the combination,WOG-alone,and low-dose PTX-alone groups,respectively.Notably,WOG combined with CDDP displayed very high toxicity.Conclusions：A synergistic inhibitory effect on growth was observed when WOG was combined with low-dose PTX in gastric cancer cells and tumor xenografts.These findings provide evidence for the design of a clinical trial to test the combination of WOG with low-dose PTX in human gastric cancer.     

Temozolomide and irradiation combined treatment-induced Nrf2 activation increases chemoradiation sensitivity in human glioblastoma cells

Resistance to chemoradiotherapy is a major obstacle to successful treatment of glioblastoma. Recently, the role of NF-E2-related factor 2 (Nrf2) in enhancing chemoradiation sensitivity has been reported in several types of cancers. Here, we investigated whether temozolomide (TMZ) and irradiation (IR) combined treatment induced Nrf2 activation in human glioblastoma cells. And we further performed a preliminary study about the effect of Nrf2 on chemoradiation sensitivity. Immunohistochemical staining for Nrf2 in paired clinical specimens showed that TMZ and IR combined treatment increased the expression and nuclear localization of Nrf2 in human glioblastoma tissues. Moreover, we found nuclear Nrf2 expression in the glioblastoma tissues obtained from the patients undergoing TMZ and IR combined treatment was associated with the time to tumor recurrence. In vitro, we further verified these findings. First, we detected increased nuclear localization of Nrf2 following treatment with TMZ+IR in human glioblastoma cell lines. Second, we demonstrated TMZ+IR increased the levels of Nrf2 protein in both nuclear and cytoplasmic fractions of U251 cells and induced Nrf2 target genes expression. Finally, downregulating Nrf2 expression increased TMZ+IR-induced cell death in the U251 cells. These findings suggest TMZ+IR combined treatment induces Nrf2 activation in human glioblastoma cells. The activation of Nrf2 may be associate with enhancing chemoradiation sensitivity in human glioblastoma cell. Blocking Nrf2 activation may be a promising method enhancing chemoradiation sensitivity of glioblastoma cells.     

Pharmaceutical-mediated inactivation of p53 sensitizes U87MG glioma cells to BCNU and temozolomide

Pifithrin-alpha (PFTalpha) is a small molecule inhibitor of p53. By reversibly blocking apoptosis in response to DNA damage, PFTalpha protects normal cells from lethal doses of gamma-radiation (Komarov et al., Science, 1999;285:1733-7). We examined the effect of PFTalpha on the chemosensitivity of a human cancer in which cell cycle arrest, not apoptosis, is the principle cellular consequence of p53 activation. This was of interest because E6 silencing of p53 sensitizes U87MG astrocytic glioma cells to BCNU and temozolomide (TMZ), cytotoxic drugs that are modestly helpful in the treatment of aggressive astrocytic gliomas. We observed that exposure of U87MG cells to PFTalpha before cytotoxic chemotherapy attenuated p53-mediated induction of p21WAF1 protein levels, sensitizing U87MG cells to BCNU and TMZ. Sensitization of U87MG cells was associated with G1 arrest, delayed entry into S-phase and decreased repair of DNA damage by BCNU. Our findings suggest that in addition to protecting normal cells from the toxic effects of radiation and chemotherapy, small molecule inhibitors of p53, like PFTalpha, might play a role in clinical oncology by sensitizing certain resistant cancers to cytotoxic chemotherapies.     

Suppression of PTEN expression is essential for antiapoptosis and cellular transformation by oncogenic Ras

Ras is one of the most commonly mutated oncogenes in the array of human cancers. The mechanism by which Ras induces cellular transformation is, however, not fully elucidated. We present here evidence that oncogenic Ras suppresses the expression of the tumor suppressor phosphatase and tensin homologue deleted from chromosome 10 (PTEN), and this action of oncogenic Ras is mediated by the Raf-mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase (MEK)-ERK pathway via up-regulation of c-Jun. Jun(+/+) cells undergo cellular transformation by oncogenic Ras, and restoration of wild-type PTEN, but not a phosphate-defective mutant of PTEN, induces apoptosis in these cells. Conversely, in Jun(-/-) cells, oncogenic Ras neither suppresses PTEN nor causes transformation, but rather it induces PTEN-dependent apoptosis. An apoptotic response to oncogenic Ras in Jun(-/-) cells can be prevented by suppressing PTEN expression. These findings imply that oncogenic Ras suppresses the apoptotic gene PTEN via the Raf-MEK-ERK-c-Jun pathway to induce antiapoptosis and cellular transformation. Together, our findings identify a novel molecular interface between the oncogenic and tumor suppressor pathways that regulates cellular transformation and survival.     

Fluosol-DA/carbogen with lonidamine or pentoxifylline as modulators of alkylating agents in the FSaIIC fibrosarcoma

Summary In an effort to increase the efficacy of several antineoplastic alkylating agents (CDDP,l-PAM, CTX, or BCNU), we examined the effect of the modulator Fluosol-DA/carbogen in combination with a second modulator, either lonidamine or pentoxifylline, on the survival of FSaIIC tumor cells and of bone marrow CFU-GM from tumor-bearing C3H mice. Fluosol-DA/carbogen increased the tumor-cell killing activity of each alkylating agent by about 10 times. In contrast, lonidamine alone did not significantly increase the cytocidal activity of any of the alkylating agents tested. However, in combination with Fluosol-DA/carbogen, the use of lonidamine produced about a 100-fold increase in the tumor cell kill achieved with CDDP as compared with CDDP alone. No increase in tumor cell kill over that produced with the single modulator Fluosol-DA/carbogen was seen following the addition of lonidamine to the combination treatment withl-PAM, CTX, or BCNU. Unfortunately, although neither lonidamine nor Fluosol-DA/carbogen alone significantly increased alkylator toxicity to bone marrow CFU-GM, the combination of modulators increased the toxicity of each alkylating agent to bone marrow by about 10 times. Pentoxifylline caused an increase in alkylator activity against the FSaIIC fibrosarcoma only when used with BCNU; this effect was further augmented by the addition of Fluosol-DA/carbogen. The combination of modulators pentoxifylline plus Fluosol-DA/carbogen was more effective than Fluosol-DA/carbogen alone only when the former was used with BCNU, whereas only minimal increases in tumor-cell killing activity were obtained with this modulator combination and CDDP,l-PAM, or CTX. Pentoxifylline increased the bone marrow CFU-GM toxicity ofl-PAM by about 10 times. The bone marrow CFU-GM toxicity was further increased by Fluosol-DA/carbogen, as was the toxicity of each of the other alkylating agents. Lonidamine plus Fluosol-DA/carbogen may be useful in increasing the therapeutic efficacy of CDDP, and the combination of pentoxifylline plus Fluosol-DA/carbogen might improve the antitumor activity of BCNU.Abbreviations CDDP cis-diamminedichloroplatinum(II) - l-PAM l-phenylalanine mustard, melphalan - thioTEPA N,N,N-triethylenethio-phosphoramide - CTX cyclophosphamide - BCNU N,N-bis(2-chloroethyl)-N-nitrosourea - CFU-GM granulocyte-macrophage colony-forming unit - MEM alpha minimal essential medium - PBS phosphatebuffered saline This work was supported by National Cancer Institute grant IPO1-CA38493, by a grant from DeSanctis Consultants, Montreal, Canada, and by a grant from the Mathers Foundation     

Expression of kinase suppressor of Ras1 enhances cisplatin-induced extracellular signal-regulated kinase activation and cisplatin sensitivity

Kinase suppressor of Ras1 (KSR1) interacts with several mitogen-activated protein (MAP) kinase pathway components, including Raf, MAP/extracellular signal-regulated kinase (ERK) kinase (MEK), and ERK, and acts as a positive regulator of the Ras signaling cascade. Previous studies have shown that exposure of cells to the anticancer agent cisplatin (cis-diamminedichloroplatinum, CDDP) is associated with changes in multiple signal transduction pathways, including c-Jun-NH2-kinase, ERK, and p38 pathways. Moreover, ERK activation has been linked to changes in cell survival following CDDP treatment. In this report, we have examined the effects of KSR1 expression on the sensitivity of cells to CDDP-induced apoptosis. Loss of KSR1 expression in mouse embryo fibroblasts (MEFs) derived from KSR1 knockout mice (KSR-/- MEF) is associated with decreased CDDP-induced ERK activation and increased resistance to CDDP-induced apoptosis compared with wild-type MEFs (KSR+/+ MEF). Furthermore, transduction of KSR-/- MEFs and MCF-7 breast cancer cells with wild-type KSR1 resulted in enhanced ERK activation following CDDP exposure and increased sensitivity to CDDP. In addition, inhibition of ERK activation by exposing MEFs to the MEK1/2-specific inhibitors PD98059 and U0126 protected both KSR+/+ and KSR-/- MEFs cells from CDDP-induced apoptosis. These results indicate that KSR1-mediated regulation of ERK activity represents a novel determinant of CDDP sensitivity of cancer cells.     

Synergy between methionine stress and chemotherapy in the treatment of brain tumor xenografts in athymic mice

This study describes a novel approach to the treatment of brain tumors with the combination of recombinant L-methionine-alpha-deamino-gamma-lyase and chemotherapeutic regimens that are currently used against such tumors. The growth of Daoy, SWB77, and D-54 xenografts in athymic mice was arrested after the depletion of mouse plasma methionine (MET) with a combination of a MET- and choline-free diet and recombinant L-methionine-alpha-deamino-gamma-lyase. The treated tumor-bearing mice were rescued from the toxic effects of MET withdrawal with daily i.p. homocystine. This regimen suppressed plasma MET to levels below 5 microM for several days, with no treatment-related deaths. MET depletion for 10-12 days induced mitotic and cell cycle arrest, apoptotic death, and widespread necrosis in tumors but did not prevent tumor regrowth after cessation of the regimen. However, when a single dose of 35 mg/m(2) of N,N'-bis(2-chloroethyl)-N-nitrosourea (BCNU), which was otherwise ineffective as a single therapy in any of the tumors tested, was given at the end of the MET depletion regimen, a more than 80-day growth delay was observed for Daoy and D-54, whereas the growth of SWB77 was delayed by 20 days. MET-depleting regimens also trebled the efficacy of temozolomide (TMZ) against SWB77 when TMZ was given to animals as a single dose of 180 mg/m(2) at the end of a 10-day period of MET depletion. The enhanced responses of both Daoy and SWB77 to DNA alkylating agents such as BCNU and TMZ could be attributed to the down-regulation of O(6)-methylguanine-DNA methyltransferase activity. However, the synergy of MET depletion and BCNU observed with D-54 tumors, which do not express measurable O(6)-methylguanine-DNA methyltransferase protein, is probably mediated by a different mechanism. MET depletion specifically sensitizes tumors to alkylating agents and does not significantly lower the toxicity of either BCNU or TMZ for the host. In this regard, the combination approach of MET depletion and genotoxic chemotherapy demonstrates significant promise for clinical evaluation.