Synergistic antileukemic effect of two polyamine synthesis inhibitors. Host survival and cell-cycle kinetic analysis

alpha-Difluoromethylornithine (DFMO), an enzyme-activated irreversible inhibitor of ornithine decarboxylase, was used alone and in combination with multiple doses of methylglyoxal-bis(guanylhydrazone) (MGBG) to treat mice with systemic L1210 leukemia. Used as a single agent (administered p.o. as a 3% solution in tap water), DFMO exerted a weak therapeutic effect against this tumor. The therapeutic effect of MGBG (administered i.p. at 50 mg/kg/day) was only slightly better. However, 1-3 days of pretreatment with DFMO strongly potentiated the effect of MGBG treatment. Thus, mice treated with the combination exhibited an increase in life span of up to 138%. The prolonged survival of leukemic mice treated with a combination of DFMO and MGBG was associated with inhibition of polyamine synthesis and a marked decrease in the spermidine and spermine content of the tumor cells as compared to untreated controls. As a consequence, there was a continuous decrease in the S- and G2-phase fractions with a concomitant increase in G1. Used singly, DFMO and MGBG had no significant effect on the cell-cycle distribution. The effects of the combination of DFMO and MGBG on the cell-cycle distribution are consistent with the contention that polyamine deficiency primarily interferes with initiation of DNA synthesis. However, the possibility that selective S-phase kill partly contributes to this change in cell-cycle distribution cannot be excluded.     

The role of angiogenesis inhibitors in prostate cancer

Prostate cancer is the leading noncutaneous malignancy in American men. Only the combination of docetaxel and prednisone has been shown to improve survival in patients with metastatic castrate-resistant prostate cancer. However, responses are short and a search for better agents either alone or synergistic with chemotherapy continues to be an urgent medical need. Angiogenesis has been shown to be a prerequisite event for tumor growth and metastasis in prostate cancer. Several strategies have been used to target angiogenesis in prostate cancer. These include blocking of pro-angiogenic factors via monoclonal antibodies or small molecule inhibitors targeting downstream signaling effector pathways, direct inhibition of endothelial cells, or targeting other receptors involved in cell adhesion, proliferation, and survival. Agents such as thalidomide and bevacizumab have shown encouraging results in phase II trials, and this review focuses on the clinical trials that have used these agents and other novel agents. The use of angiogenesis inhibitors is rapidly emerging as a promising treatment strategy in a variety of solid tumors, currently including prostate cancer.     

Sequential inhibition of polyamine synthesis

Both DFMO and methyl-GAG inhibit sequential enzymatic reactions in the pathway of polyamine biosynthesis. Since polyamines may be important factors in proliferation of cancer cells, we initiated a phase-I study of these agents in patients with advanced cancer. DFMO was given by mouth at a constant daily dose of 4 g/m2 starting on day 1 of the treatment protocol. The dose of methyl-GAG ranged from 200 to 700 mg/m2 administered IV every 2 weeks beginning on day 4. Twenty-two patients were entered into the protocol. Toxic reactions to this therapy were dose-related and included nausea, fatigue, diarrhea, and myelosuppression. One patient with colon cancer experienced a greater than 50% decrease in measurable disease but developed severe myelotoxicity. While DFMO was well tolerated, the combination of drugs appeared to cause substantially more hematologic and gastrointestinal toxicity than encountered during our recent experience with methyl-GAG used alone. We suggest that future studies of this drug combination carefully evaluate levels of polyamines and inhibition of enzymatic activity to minimize toxicity.     

Low-deuterium water effect on transplantable tumors

Such models of transplantable tumors as Lewis sarcoma, uterine sarcoma-322 and uterine carcinoma-5 were used to study possible inhibitory effect by low-deuterium water. Such water was given to experimental animals (20 mice in each group). Controls (30 in each group) received tap water with standard deuterium concentrations. Low-deuterium water treatment resulted in significant inhibitory effect on volume of all tumor patterns concerned: it delayed nodule formation at transplantation site. However, no increase in survival time was obtained.     

Genes in the polyamine biosynthesis pathway may be involved in prostate cancer susceptibility

One of the most investigated low-penetrance genes is the androgen receptor gene. A recent meta-analysis showed however that the absolute difference in number of repeats between cases and controls was less than one repeat. This result has questioned whether the androgen receptor gene could be functionally important in prostate cancer etiology. The authors hypothesize that genes that are downstream from the androgen receptor gene, potentially those involved in testosterone response, could be of more interest. One of the primary responses of prostate cells to testosterone is the production of polyamines. Recently, a meta-analysis across gene-expression profiling studies found that genes in the polyamine biosynthesis pathway appear to be consistently dysregulated in prostate cancer. Polyamines are also involved in prostate diagnosis and treatment. Therefore, the authors suggest that future oncologic research to identify candidate regions for prostate cancer should focus on genes dysregulated in this pathway.     

Effect of intravenous alpha-difluoromethylornithine on the polyamine levels of normal tissue and a transplantable fibrosarcoma

The effect of a continuous i.v. infusion of alpha-difluoromethylornithine (DFMO) on the polyamine metabolism of tumor and normal host tissue was determined. Non-tumor-bearing Fischer 344 rats or rats bearing a transplantable fibrosarcoma received continuous infusions of DFMO through a central venous catheter at three dose levels. Treatment with DFMO resulted in a time- and dose-dependent, cytostatic effect on the growth of the tumor. In fibrosarcoma-bearing rats the tumor putrescine levels were reduced after 6 and 12 days of DFMO treatment. Tumor spermidine levels were consistently reduced after 6 and 12 days of treatment with the reduction being dose dependent. The decrease in tumor ornithine decarboxylase activity was dose dependent. Erythrocyte putrescine levels were decreased in tumor- and non-tumor-bearing rats, suggesting that DFMO reduces the tumor contribution to the erythrocyte pool. Erythrocyte spermidine levels of fibrosarcoma- and non-tumor-bearing rats were elevated at the lower DFMO doses administered for 12 days but returned to normal as the dose was increased. Erythrocyte spermine levels were elevated in both groups of rats at all DFMO doses. Although normal host tissue weights were not affected by treatment with DFMO, the putrescine and spermidine levels of liver, spleen, and kidney and ornithine decarboxylase activity of the liver and kidney were decreased. These data demonstrate that i.v. DFMO has a cytostatic effect toward a rapidly growing fibrosarcoma associated with the depletion of both tumor putrescine and spermidine levels.     

Relationship of erythrocyte polyamine levels and growth rate of transplantable tumors in rats

Varying levels of polyamines in the urine, plasma, and erythrocytes (RBC) of cancer patients have been demonstrated. The growth rate of the tumor has been suggested as a primary factor which determines whether the polyamine levels in urine are elevated. To further evaluate tumor size and growth rate as variables affecting polyamine levels in physiological fluids, the effect of a transplantable fibrosarcoma and colon tumor on the RBC polyamine levels of Fischer 344 rats was determined. The tumors were implanted s.c. and grew without metastasis or spontaneous regression. The fibrosarcoma grew exponentially up to a weight of approximately 69 +/- 15 (SD) g and was associated with a linear increase in RBC polyamine levels compared with that of non-tumor-bearing rats. RBC putrescine, spermidine, and spermine levels were significantly elevated at tumor weights of 12.5 +/- 1.4, 20.4 +/- 3.8, and 33.2 +/- 5.0 g, respectively. The respective polyamines increased consistently thereafter until the tumor weight was 57.8 +/- 5.8 g. In contrast with the fibrosarcoma, the colon tumor grew exponentially only to a weight of 9.2 +/- 4.7 g, at which time the growth rate of the tumor began to decrease (time T of the Gompertz model). RBC polyamine levels of rats with the colon tumor showed only a transient increase. RBC putrescine levels were significantly increased at a tumor weight of 12.9 +/- 1.2 g and spermidine at a tumor weight of 17.4 +/- 0.2 g. RBC spermine levels were significantly elevated at both tumor weights; thereafter, all RBC polyamine levels returned to normal. Host cachexia was evident when the fibrosarcoma and colon tumors weighed 12.5 +/- 0.9 and 7.2 +/- 2.6 g, respectively. The polyamine levels of the fibrosarcoma differed significantly from that of the colon tumor. These levels, however, did not correlate with the exponential growth rates. The results suggest that the tumor is the major source of elevated RBC polyamines. The data also suggest that the tumors must be rapidly growing for the elevation in polyamines to occur. This may partly explain why patients with extensive neoplastic disease that may have surpassed time T in the Gompertz model do not manifest abnormal polyamine levels.     

姜黄素联合肿瘤坏死因子相关凋亡诱导配体对前列腺癌细胞的抑制作用

目的:探讨姜黄素(curcumin,Cur)联合肿瘤坏死因子相关凋亡诱导配体(TNF-related apoptosis-inducing ligand,TRAIL)对前列腺癌细胞的抑制作用。方法:CCK-8法测定Cur、TRAIL及Cur联合TRAIL作用于PC-3细胞后的细胞生存率,流式细胞技术测定细胞周期及凋亡率,光学显微镜下观察细胞形态,Western blot检测凋亡蛋白caspase-3表达水平的变化。结果:与对照组比较,Cur联合TRAIL组能显著抑制PC-3细胞增殖(P<0.05),呈时间依赖性,且使PC-3细胞阻滞于G2/M期比例明显增多(P<0.05)。镜下可见联合应用Cur和TRAIL处理的PC-3细胞凋亡形态改变明显,凋亡率较对照组高(P<0.05),凋亡蛋白caspase-3表达也增强。结论:Cur与TRAIL联用可明显增强PC-3细胞抑制效果和诱导凋亡作用,其凋亡作用机制可能与其上调caspase-3表达有关。     

Study of matrix metalloproteinases and their inhibitors in prostate cancer

Background:

Extracellular matrix metalloproteases (MMPs) have raised an extraordinary interest in cancer research because of their potential role in basal membrane and extracellular matrix degradation, consequently facilitating tumour invasion and metastases development.

Methods:

An immunohistochemical study was performed using tissue arrays and specific antibodies against MMPs 1, 2, 7, 9, 11, 13, 14, and their tissue inhibitors, TIMPs 1, 2 and 3. More than 2600 determinations on cancer specimens from 133 patients with clinically localised prostate carcinoma, 20 patients with prostatic intraepithelial neoplasia and 50 patients with benign prostate hyperplasia and controls, were performed.

Results:

When compared with benign pathologies, prostate carcinomas had higher expression of all MMPs and TIMPs. Dendogram shows a first-order division of tumours into two distinct MMPs/TIMPs molecular profiles, one of them with high MMPs/TIMs expression profile (n=70; 52.6%). Tumours with high expression of MMP-11 or -13, or cluster thereof, were significantly associated with higher probability of biochemical recurrence.

Conclusion:

The expression of MMPs and TIMPs seems to have an important role in the molecular biology of prostate carcinomas, and their expression by tumours may be of clinical interest to used as indicators of tumour aggressiveness.     

The effect of dose on local control of prostate cancer

Three patterns of care outcome surveys in prostate cancer totalling 1516 patients had been combined and analyzed for the effect of dose on infield recurrence. There are significant dose effects observed in the overall data (1516 patients, p = .003), Stage B cancers (725 patients, p = .004) and Stage C cancers (624 patients, p = .059). No dose effect was observed for Stage A cancers (168 patients, p = .217) within the dose range observed (5500 cGy to greater than 7000 cGy). For patients with Stage B cancer one may conclude that dose between 6000 cGy and 6999 cGy is appropriate. Patients treated to less than 6000 cGy show a highly significant increase in local failure. Patients treated to greater than 7000 cGy do not show a demonstrable improvement in local control, but do show an increase in complications. Patients with Stage C cancer appear to require dose that is equal or greater than 7000 cGy to obtain the best local control, and the potential increased morbidity of these high doses appears to be justified in this stage of the disease. Patients who have been given hormonal therapy more than 1 month prior to radiation therapy show an increase in local failure rate for all stages of cancer. This is presumed to be the selection of poor risk patients for adjuvant hormonal treatment or by referring non-responding hormone treated patients for radiation therapy. Histologic grade exerts a major influence on local failure for patients with Stage C disease (p = less than .001), identifying an important stratification point for prospective clinical trials and a sub-group for which it is important to develop strategies for improving local control. The policy of treating all stages of prostate cancer with the same dose is not supported by these data.     

浅蓝菌素对前列腺癌细胞株PC-3生长抑制的研究

目的:探讨脂肪酸合成酶(FAS)的抑制剂浅蓝菌素对前列腺癌细胞株PC-3的生长抑制作用。方法:采用人前列腺癌细胞株PC-3在体外实验中用MTT法观察浅蓝菌素对细胞株的生长抑制作用;流式细胞术(FCM)对细胞株的凋亡及细胞周期的变化进行检测;蛋白免疫印迹(W estern b lotting)法检测浅蓝菌素对PC-3细胞中FAS蛋白水平表达的影响。结果:PC-3细胞在浅蓝菌素的作用下,细胞增殖被明显阻遏,并呈现剂量效应关系,在浓度为2.5mg/L、5.0mg/L、10.0mg/L、20.0mg/L的浅蓝菌素作用下,PC-3细胞24h的抑制率分别为(34.78±2.47)%、(46.76±3.18)%、(58.13±3.55)%、(65.31±2.81)%,与对照组比较有统计学意义(P<0.05)。流式细胞仪显示,细胞经浅蓝菌素作用后,细胞凋亡增多,PC-3细胞在浅蓝菌素浓度5.0mg/L三个时间组(24h,48h,72h)的细胞凋亡率分别为(28.29±1.88)%、(44.73±2.69)%、(61.25±1.43)%;且G0/G1期细胞比例增加,S期和G2/M期细胞比例减少。蛋白免疫印迹实验中,随着浅蓝菌素作用时间的延长,PC-3细胞中的FAS蛋白水平表达量明显减少,药物作用时间与细胞中蛋白水平表达量呈负相关。结论:浅蓝菌素能抑制PC-3细胞生长,且其呈现时间和浓度依赖性。     

浅蓝菌素对前列腺癌细胞株PC-3生长抑制的研究

目的：探讨脂肪酸合成酶（FAS）的抑制剂浅蓝菌素对前列腺癌细胞株PC-3的生长抑制作用。方法：采用人前列腺癌细胞株PC-3在体外实验中用MTT法观察浅蓝菌素对细胞株的生长抑制作用;流式细胞术（FCM）对细胞株的凋亡及细胞周期的变化进行检测;蛋白免疫印迹（W estern b lotting）法检测浅蓝菌素对PC-3细胞中FAS蛋白水平表达的影响。结果：PC-3细胞在浅蓝菌素的作用下,细胞增殖被明显阻遏,并呈现剂量效应关系,在浓度为2.5mg/L、5.0mg/L、10.0mg/L、20.0mg/L的浅蓝菌素作用下,PC-3细胞24h的抑制率分别为（34.78±2.47）%、（46.76±3.18）%、（58.13±3.55）%、（65.31±2.81）%,与对照组比较有统计学意义（P〈0.05）。流式细胞仪显示,细胞经浅蓝菌素作用后,细胞凋亡增多,PC-3细胞在浅蓝菌素浓度5.0mg/L三个时间组（24h,48h,72h）的细胞凋亡率分别为（28.29±1.88）%、（44.73±2.69）%、（61.25±1.43）%;且G0/G1期细胞比例增加,S期和G2/M期细胞比例减少。蛋白免疫印迹实验中,随着浅蓝菌素作用时间的延长,PC-3细胞中的FAS蛋白水平表达量明显减少,药物作用时间与细胞中蛋白水平表达量呈负相关。结论：浅蓝菌素能抑制PC-3细胞生长,且其呈现时间和浓度依赖性。     

血红素加氧酶-1在前列腺癌中的表达

目的探讨血红素加氧酶-1(HO-1)在人前列腺癌组织中的表达,以及HO-1活性对体外培养的前列腺癌细胞(PC3)的影响。方法　收集我院2000年1月至2007年4月前列腺癌根治性手术标本30例,前列腺增生开放手术大体标本30例。行RT-PCR检测HO-1的mRNA表达,免疫组化检测HO-1的表达。应用ZnPP、CoPP干预体外培养的PC3细胞,24h后,RT-PCR检测HO-1 mRNA的表达,MTT法酶标检测仪检测细胞A570nm值,流式细胞术检测细胞凋亡。结果人前列腺癌组织HO-1的mRNA表达明显高于良性增生前列腺组织;免疫组化前列腺癌中HO-1表达阳性率为76.9%,良性前列腺组织仅少许有微弱表达。应用ZnPP、CoPP干预PC3细胞后,RT-PCR可见ZnPP抑制PC3细胞HO-1表达,且随浓度增加抑制作用增加;CoPP可诱导HO-1表达,随浓度增加表达增强。MTT检测显示,ZnPP组PC3细胞A值随浓度增加而减少,而CoPP组随浓度增加A值增加,呈现浓度依赖性(P<0.05)。流式细胞技术结果显示,ZnPP可诱导PC3细胞凋亡,CoPP可保护细胞减少凋亡。结论人前列腺癌组织中HO-1基因表达及蛋白水平均显著高于良性前列腺组织;体外细胞实验证实,ZnPP可抑制前列腺癌PC3细胞系HO-1活性,抑制其增殖,诱导其凋亡。而CoPP可诱导前列腺癌PC3细胞系HO-1表达,抑制其凋亡,诱导其增殖。