姜黄素抑制肾癌ACHN细胞增殖及促凋亡的实验研究

目的观察姜黄素对人肾癌ACHN细胞株增殖及细胞凋亡的影响,探讨姜黄素诱导ACHN细胞株凋亡的作用机制。方法不同浓度姜黄素作用人肾癌ACHN细胞24 h后,应用MTT比色法检测姜黄素对人肾癌ACHN细胞的增殖抑制率;流式细胞术检测姜黄素诱导细胞的凋亡率;RT-PCR检测姜黄素对ACHN细胞Bcl-2、Bax、NF-κBP65 mRNA表达的影响;Western blot方法检测其对细胞Bcl-2、Bax、NF-κBP65I、κB蛋白表达的影响。结果姜黄素对人肾癌ACHN细胞有明显的抑制作用,可引起细胞凋亡,并且存在剂量和时间依赖;不同浓度姜黄素作用细胞24 h后,Bcl-2、NF-κBP65 mRNA水平下降,Bax mRNA水平升高(P0.05),Bcl-2、NF-κBP65蛋白表达量下降,BaxI、κB蛋白表达量升高(P0.05)。结论姜黄素通过上调IκB,下调NF-κB活性,调控凋亡基因Bcl-2/Bax的表达,抑制人肾癌ACHN细胞的增殖,诱导人肾癌ACHN细胞的凋亡。     

Flavopiridol对尤文肉瘤SK-N-MC细胞增殖的抑制作用

目的探讨细胞周期蛋白激酶抑制剂（flavopiridol）在体内外抑制尤文肉瘤细胞增殖的作用及其机制。方法以不同浓度的flavopiridol作用于培养的SK-N-MC细胞,用噻唑蓝法（MTT）检测细胞增殖,流式细胞分析仪测定细胞周期。采用DNA凝胶电泳检测法,观察flavopiridol诱导细胞凋亡的情况;蛋白质电泳观察Bcl-2及Mcl-1表达变化;建立SK-N-MC细胞裸小鼠皮下移植瘤模型,腹腔内给药,观察肿瘤生长情况;肿瘤标本石蜡包埋切片,免疫组织化学原位末端标记法（TUNEL）检测flavopiridol在体内对肿瘤细胞的凋亡诱导情况。结果（1）Flavopiridol对人尤文肉瘤细胞增殖有抑制作用,时间越长、浓度越高,抑制作用越强。（2）Flavopiridol可改变尤文肉瘤细胞周期,随药物浓度增加Sub-G1期百分比逐渐上升。尤文肉瘤细胞经flavopiridol中、高浓度给药后,DNA凝胶电泳可见典型的梯状带。（3）Flavopiridol给药前后,Bcl-2蛋白表达无变化,而Mcl-1蛋白表达明显下降。（4）TUNEL染色结果显示实验组阳性细胞比例上升,与对照组比较差异有统计学意义。结论Flavopiridol能在体内外抑制尤文肉瘤细胞的增殖,其机制主要是阻滞细胞周期的G1/S期,诱导细胞凋亡。     

Over expression of inhibitor of caspase 3 activated deoxyribonuclease in human renal cell carcinoma cells enhances their resistance to cytotoxic chemotherapy in vivo.

PURPOSE: We determined whether inhibitor of caspase-3 activated deoxyribonuclease (ICAD) enhances resistance to apoptotic stimuli or inhibits DNA fragmentation by inactivating caspase-3 activated deoxyribonuclease. MATERIALS AND METHODS: The liposome mediated gene transfer method was used to introduce ICAD complementary DNA into ACHN cells and the expression of ICAD protein per clone was evaluated by Western blot analysis. Effects of cisplatin treatment on growth inhibition and apoptosis in the ACHN sublines were assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, DNA fragmentation assay and Western blot analysis of poly(ADP-ribose) polymerase protein. The limiting dilution assay was also performed to examine the effect of cisplatin treatment under anchorage independent conditions. Furthermore, nude mice bearing the ACHN sublines were given intraperitoneal injection of 5 mg./kg. cisplatin 1 and 2 weeks after the implantation of tumor cells and changes in tumor volume was measured. RESULTS: ICAD transfected ACHN cells inhibited DNA degradation after cisplatin treatment but not control vector only transfected ACHN cells, whereas a similar degree of apoptotic cell death induced by cisplatin in ICAD and control vector only transfected ACHN cells was observed on 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and Western blot analysis of poly(ADP-ribose) polymerase protein. However, the limiting dilution assay revealed that ICAD transfected ACHN cells had high resistance to pretreatment with cisplatin compared with control vector only transfected ACHN cells. Moreover, although there was no significant difference in the in vivo growth of ACHN sublines, cisplatin treatment induced the elimination of control vector only transfected ACHN cell tumors. In contrast, most ICAD transfected ACHN cell tumors continued to grow after cisplatin treatment. CONCLUSIONS: These findings suggest that when ICAD is over expressed in tumor cells, it renders them highly resistant to therapy induced apoptosis, resulting in enhanced tumor progression.     

Synergistic antitumor effects of HER2/neu antisense oligodeoxynucleotides and conventional chemotherapeutic agents.

BACKGROUND: The HER2/neu oncogene is overexpressed in a substantial fraction of human tumors. HER2/neu overexpressing tumors may be intrinsically resistant to chemotherapy. The present study examined the ability of antisense-mediated downregulation of HER2/neu expression to enhance the antitumor effects of conventional chemotherapeutic agents against human tumor cells that overexpress HER2/neu. METHODS: The effects of HER2/neu antisense oligodeoxynucleotides (ODNs) on the growth inhibitory and proapoptotic activity of several distinct chemotherapeutic agents were examined in vitro. In vivo effects of HER2/neu antisense ODNs in combination with doxorubicin hydrochloride were assessed by examining the growth of human tumor xenografts implanted into nude mice. RESULTS: The proliferation of tumor cell lines that overexpress HER2/neu was inhibited by antisense ODNs in combination with conventional chemotherapeutic agents in an additive or synergistic fashion. Such combination therapy also demonstrated synergistic activation of apoptosis. HER2/neu antisense ODNs in combination with doxorubicin hydrochloride demonstrated synergistic antitumor effects in vivo as well. CONCLUSIONS: Downregulation of HER2/neu expression can enhance the sensitivity of human cancer cells, which overexpress HER2/neu to the cytotoxic effects of chemotherapy. Antisense ODNs targeting the HER2/neu gene may play a role in cancer therapy.     

Introducing the clusterin gene into human renal cell carcinoma cells enhances their metastatic potential

PURPOSE: We recently reported a protective role of clusterin expression against apoptosis induced by a wide variety of stimuli in several human cancer models. In the current study we tested the hypothesis that clusterin over expression confers a benefit for the metastasis of renal cell carcinoma through the inhibition of apoptosis induced by the various obstacles the cancer cells may confront after detachment from their primary origin. MATERIALS AND METHODS: We introduced clusterin complementary DNA into human renal cell carcinoma ACHN cells, which do not express detectable level of clusterin expression, and generated the clusterin over expressing cell line ACHN/CL and the control vector only transfected cell line ACHN/C. In vitro anti-cell death activity under anchorage independent conditions among ACHN sublines was examined by limiting dilution assay and cell survival assay in suspension. To investigate the in vivo effects of clusterin over expression on metastatic potentials each cell line was injected into the tail vein or renal subcapsule of nonobese diabetic, severe combined immunodeficient mice and the metastatic features in all abdominal and thoracic organs were evaluated. RESULTS: ACHN/CL showed significantly enhanced growth in limiting dilution cultures compared with ACHN/C. The analysis of cell survival in the floating assay also revealed that ACHN/CL had a powerful survival advantage in suspension compared with ACHN/C. Furthermore, ACHN/CL formed more than 5-fold as many metastatic nodules in the lung after intravenous injection than ACHN/C. Similarly more marked lung metastasis was observed after implanting ACHN/CL cells into the renal subcapsule than after implanting ACHN/C cells. In contrast, there were no significant differences among ACHN sublines in the growth rates in vitro and in vivo, cell motility or invasive ability. CONCLUSIONS: These findings suggest that, if clusterin is over expressed, it prolongs cell survival under unfavorable conditions in the metastatic process, resulting in the enhanced metastatic potential of renal cell carcinoma.     

内皮抑素基因对ACHN RCC中VEGF和MVD表达的影响

目的：利用已构建的带有内皮抑素基因真核表达载体质粒（pSecES）导入裸鼠人肾癌细胞系肾细胞癌（ACHN RCC）中,研究内皮抑素基因对血管内皮生长因子（VEGF）和微血管密度（MVD）表达的影响.方法：将荷瘤裸鼠随机分为3组,每组8只.干预组每只裸鼠瘤内3点注射复合物100μl,内含30μl梭华-Sofasttm和30μg pSecES质粒;对照组每只裸鼠瘤内3点注射复合物100μl,内含30μl梭华-Sofasttm和30μg pcDNA3.1质粒;空白组每只裸鼠瘤内3点注射生理盐水100μl.各组每只裸鼠间隔3天注射1次,连续注射3次.接种后第34天处死裸鼠,取肿瘤组织按SP法进行免疫组织化学染色,检测MVD和VEGF的表达情况.结果：干预组ACHN RCC的生长较对照组和空白组明显受到抑制,干预组CD34标记的MVD的表达和VEGF的表达较对照组和空白组低.结论：内皮抑素基因导入可以抑制荷瘤裸鼠ACHN RCC的生长,而且MVD和VEGF在ACHNRCC的表达减少.     

Roles of NF-kappaB and bcl-2 in two differential modes of cell death of mouse cortical collecting duct cells

Recent data have implicated nuclear factor-kappaB (NF-kappaB) and Bcl-2 in the regulation of apoptotic and necrotic cell death in various cells. However, mechanisms of their effects on cell death of renal epithelial cells are not clear. First, we investigated the effect of specific inhibition of NF-kappaB and overexpression of Bcl-2 on necrotic cell death induced by hydrogen peroxide or cisplatin in renal collecting duct cells. M-1 cells, which were derived from outer cortical collecting duct, were stably transfected with the non-phosphorylatable mutant of inhibitory-kappaBalpha (I-kappaBalpha) and Bcl-2. Overexpression of I-kappaBalpha and Bcl-2 did not affect cisplatin-induced necrotic cell death, but overexpression of I-kappaBalpha significantly decreased H2O2-induced cell death. Regarding apoptotic cell death induced by cisplatin, serum deprivation and contact inhibition was increased by overexpression of I-kappaBalpha, whereas overexpression of bcl-2 inhibited the apoptotic cell death. I-kappaBalpha overexpression increased Bax expression and decreased cIAP-1 and -2 expression compared to vector-transfected cells, but did not alter SAPK/JNK activity in the presence or absence of cisplatin. NF-kappaB activity was significantly higher in bcl-2-overexpressing cells than in control cells. These data show that activation of NF-kappaB mediates H2O2-induced necrotic injury, but inhibits apoptotic cell death in renal collecting duct cells, and that Bcl-2 selectively protects apoptotic cell death in M-1 cells.     

Antitumor Effect of Irinotecan Hydrochloride (CPT-11) on Human Renal Tumors Heterotransplanted in Nude Mice

Background : There has been a paucity of antitumor drugs that are active against renal tumors. Irinotecan hydrochloride (CPT-11), a DNA topoisomerase type 1 inhibitor, has demonstrated antitumor activity against human tumors, however, no antitumor effect of CPT-11 on renal tumors has been reported. The antitumor effect of CPT-11 was investigated on 2 human renal tumors (OUR-10 and OUR-20) heterotransplanted into nude mice.
Methods : Tumor-bearing nude mice were given daily intraperitoneal injections of multiple anticancer drugs suspended in 0.2 ml_ of phosphate-buffered saline (PBS) 3 times at 3-day intervals. Control mice were injected with 0.2 mL of PBS. The antitumor effects were evaluated by calculating the T/C ratio (treated tumors/controls) of the tumor volume.
Results : Among the 10 anticancer drugs tested, 50 mg/kg of CPT-11 showed an active antitumor effect on OUR-20 (T/C ratio 34). However, all drugs tested on OUR-10 failed to show antitumor activity. Conclusion: Since CPT-11 was effective in 1 of 2 renal tumors examined without severe toxicity, this drug could be a candidate for chemotherapy of renal cell carcinoma.     

Synergistic enhancement of resistance to cisplatin in human bladder cancer cells by overexpression of mutant-type p53 and Bcl-2

PURPOSE: The objective of this study was to characterize the effect of mutant-type p53 and Bcl-2 expression on the sensitivity to cisplatin in a human bladder cancer cell line both in vitro and in vivo. MATERIALS AND METHODS: We transfected mutant-type p53 cDNA, Bcl-2 cDNA, or both cDNAs into KoTCC-1, a human bladder cancer cell line that does not express mutant-type p53 or Bcl-2 protein. The effects of the overexpression of mutant-type p53, Bcl-2, or both on the sensitivity to cisplatin and the apoptotic features in vitro were evaluated by the MTT assay, staining with Hoechst 33258 and a DNA fragmentation assay. We then examined the in vivo effects of cisplatin treatment on the transfectants by subcutaneous and intraperitoneal tumor cell injection models in athymic nude mice. RESULTS: The introduction of mutant-type p53 or Bcl-2 conferred resistance to cisplatin on KoTCC-1 cells through the inhibition of apoptosis. This phenotype was more remarkable in the cell line transfected with both mutant-type p53 and Bcl-2 than in the cell lines transfected with either mutant-type p53 or Bcl-2 alone. Furthermore, the KoTCC-1 cells transfected with both mutant-type p53 and Bcl-2 exhibited significantly higher resistance to cisplatin treatment than cells transfected with mutant-type p53 or Bcl-2 alone in experimental models in vivo. CONCLUSIONS: These findings suggest that the overexpression of both mutant-type p53 and Bcl-2 in bladder cancer cells synergistically interferes with the therapeutic effect of cisplatin through the inhibition of the apoptotic pathway.     

放射治疗对小鼠肾癌细胞凋亡和Bcl-2与Bax蛋白表达的影响

目的:观察放射治疗对BALB/c小鼠肾癌的抑制作用及对Bcl-2和Bax蛋白表达的影响.方法:建立BALB/c小鼠的肾癌细胞移植瘤放射治疗模型;HE染色测定肿瘤细胞死亡面积,采用免疫印迹法(western blot法)检测肿瘤组织的凋亡相关蛋白表达.结果:放疗组小鼠移植瘤生长速度较肿瘤对照组明显减慢.放疗组小鼠肿瘤组织中抗凋亡蛋白Bcl-2表达下调,促凋亡蛋白Bax表达上调.结论:经放射治疗后,小鼠肿瘤生长受到明显抑制,可能与放疗引发肿瘤细胞凋亡相关蛋白表达变化相关.     

反义K-ras基因对胰腺癌细胞增殖和凋亡的影响

目的 探讨反义K-ras癌基因对胰腺癌细胞增殖和凋亡及Fas/FasL、Bcl-2、Bax表达的影响.方法 将重组逆转录病毒载体pLXSN转染包装细胞PT-67,获得重组逆转录病毒.在细胞和动物水平将该重组逆转录病毒分别感染PC-3和BxPC-3胰腺癌细胞,经G418筛选获得稳定细胞系,应用MTT、流式细胞仪和免疫组化法分别研究其增殖、凋亡及其相关基因表达情况.结果 成功制备含反义K-ras基因的重组逆转录病毒.感染含反义K-ras基因重组病毒后,胰腺癌PC-3细胞和移植瘤(有K-ras基因第12密码子点突变GGT→GTT)增殖受到抑制,G<,0/1>期细胞增加而S期细胞明显减少,细胞凋亡增加.免疫组化显示含反义K-ras癌基因逆转录病毒感染后PC-3细胞Bax/Bcl-2比值升高,Fas表达上调.结论 反义K-ras基因可使胰腺癌细胞及移植瘤增殖受到抑制,并可通过上调Bax/Bcl一2比值和(或)促进Fas表达诱导细胞凋亡.     

人结肠癌裸鼠移植瘤热疗和化疗及放疗后凋亡相关基因的变化

目的探讨热疗、化疗、放疗对人结肠癌裸鼠移植瘤凋亡相关基因表达的影响。方法将人类结肠癌细胞株——HT29移植于裸鼠双侧后肢。于实验室模拟热疗条件（43℃，60rain），并参照人类结肠癌临床化疗及放疗方案。实验共分6组，即热疗、化疗、放疗、热化疗、热放疗及热放化疗组。于治疗前后不同时间点处死裸鼠。取肿瘤组织备用。利用免疫组织化学染色检测肿瘤细胞膜、细胞浆及细胞核Bcl－2和Bax基因及P53表达的形态学变化。结果6种治疗方法均能不同程度下调P53和Bcl－2、上调Bax的表达。热放化疗组治疗后48hP53及Bcl－2达到最低水平，Bax达高峰。与另外5组比较差异有统计学意义（P〈0．01）；热疗、热化疗及热放疗3组间对P53表达的下调作用差异无统计学意义，但均较化疗、放疗组下调明显。治疗后48h热放疗组和热放化疗组Bcl－2的表达明显低于其他各组（P〈0．05），热疗组下调Bcl－2作用最小，与另外5组的差异均有统计学意义（P〈0．05和P〈0．01）。热放化疗组上调Bax基因表达的作用发生时间最早（2h）：作用最强，与另外5组的差异均有统计学意义（P〈0．05和P〈0．01）；热疗组发生时间最晚（24h），作用最小，与另外5组的差异均有统计学意义（P〈0．05和P〈0．01）。结论热疗可能通过增强放化疗对凋亡相关基因的表达发挥其增敏作用。热疗联合应用化疗和（或）放疗对人结肠癌裸鼠移植瘤凋亡相关基因表达的改变更为显著。     

苦参碱对人ACHN肾癌裸鼠皮下移植瘤的生长抑制作用

目的探讨苦参碱对人ACHN肾癌裸鼠皮下模型的生长抑制作用。方法皮下包埋法建立人ACHN肾癌裸鼠皮下移植瘤模型,按照随机原则将裸鼠分为50、75、100mg/kg苦参碱组和生理盐水组,各组均采用腹腔注射给药,5次/周,连续3周。每天测量肿瘤最长径和最短经,计算肿瘤体积,比较各组肿瘤生长抑制率;光镜及电镜观察苦参碱作用后肿瘤细胞形态改变。结果 35只裸鼠成瘤27只,观察满3周后,50、75、100mg/kg苦参碱组的肿瘤生长抑制率分别为19.51%、38.73%、46.08%,光镜下见苦参碱给药组不同程度的细胞坏死,且坏死范围随苦参碱浓度的增大而增大;电镜观察发现苦参碱作用后出现典型的细胞凋亡和坏死形态表现。结论苦参碱对人ACHN肾癌具有明显的生长抑制作用,这为临床应用苦参碱治疗肾癌提供了实验基础。     

重组人白介素-24蛋白对荷人A375细胞黑素瘤裸鼠的抑瘤作用

目的 研究原核表达及纯化、复性的重组人白介素24蛋白(recombined human interlukin 24,rhIL-24)对荷人A375细胞黑素瘤裸鼠的抑制肿瘤生长的作用.方法 将人A375黑素瘤细胞注射至裸鼠体内,待肿瘤生长到一定体积后,向裸鼠的肿瘤内注射rhIL-24,2周后切取肿瘤称重、量体积,进行病理检查及免疫组化检测.结果 经rhIL-24注射的肿瘤体积及重量与对照组相比明显减小、减轻;Bax基因表达上调、Bcl-2、CD34及VEGF基因表达下调,显示肿瘤细胞生长受到抑制,凋亡率增加.结论 rhIL-24对荷人A375细胞黑素瘤裸鼠的肿瘤有显著抑制生长的功能,可促进A375细胞凋亡,而对裸鼠则无明显的不良反应.     

Novel HER2 selective tyrosine kinase inhibitor, TAK-165, inhibits bladder, kidney and androgen-independent prostate cancer in vitro and in vivo

PURPOSE: TAK-165 is a new potent inhibitor of human epidermal growth factor receptor 2 (HER2) tyrosine kinase. Several reports suggest HER2 expression in bladder cancer, renal cell carcinoma (RCC) and androgen-independent prostate cancer. We therefore investigated the antitumor effect of TAK-165 on these urological cancer cells. MATERIALS AND METHODS: Western blot analysis was performed to confirm HER2 expression in cell lines. To study in vitro efficacy, cells were treated with TAK-165 at various concentrations for 72 h and then counted using a hemocytometer. Then the IC50 value was calculated. In the xenograft model, after the tumor reached 200-300 mm3 in volume, mice were orally administered TAK-165 10 mg/kg per day or 20 mg/kg per day or saline for 14 consecutive days (n=6-8). RESULTS: HER2 expression was observed in HT1376, UMUC3, T24 (bladder), ACHN (kidney), DU145, LNCaP, LN-REC4 (prostate), although the expression level in these cells was weak compared with BT474 (a breast cancer cell line which expresses HER2 strongly). IC50 was varied from 0.09 to greater than 25 micromol/L in the bladder cancer cell line. ACHN cells were less sensitive in vitro. The prostate cancer cell lines studied were all sensitive (IC50 0.053-4.62 micromol/L). In the xenograft model, treatment with TAK-165 significantly inhibited growth of UMUC-3, ACHN, and LN-REC4. The antitumor effect (T/C [%]=growth of TAK-165 treated tumor/average growth of control tumorx100) after 14 days treatment were 22.9%, 26.0%, and 26.5% in UMUC3, ACHN and LN-REC4, respectively. CONCLUSIONS: TAK-165 may be a hopeful new agent for bladder, kidney and androgen-independent prostate cancer.     

Sunitinib malate is active against human urothelial carcinoma and enhances the activity of cisplatin in a preclinical model

PurposeSunitinib malate (Pfizer, Inc.) is a multitargeted kinase inhibitor that inhibits vascular endothelial growth factor (VEGF) receptor (R)-1, 2 and 3, platelet-derived growth factor receptors (PDGFR)-α and β, Flt3, RET, and Kit. Angiogenesis and VEGF expression correlate with poor outcomes in human urothelial carcinoma. We designed a preclinical study to examine the efficacy of sunitinib alone and in combination with cisplatin against human urothelial carcinoma.DesignThe in vitro activities of sunitinib and cisplatin alone and in combination were determined against human urothelial carcinoma cell lines, TCC-SUP and 5637. Antitumor activities were also determined in vivo against murine subcutaneous 5637 xenografts. Immunohistochemistry (IHC) was performed to detect VEGFR2 and Kit, and modulation of proliferation, apoptosis, and angiogenesis.ResultsBoth cell lines expressed VEGFR2, but did not express Kit. Sunitinib displayed activity against both cell lines in vitro at low micromolar concentrations, which are not attainable in vivo, and was synergistic with cisplatin. Activity was observed for sunitinib at 20 and 40 mg/kg orally once daily for 4 weeks, which attains low nanomolar concentrations in vivo against murine 5637 xenografts. Sunitinib 20 mg/kg/d in combination with cisplatin 4 mg/kg/wk intraperitoneally induced tumor regression compared to no therapy (P < 0.0001) or cisplatin alone (P = 0.06). Cisplatin, sunitinib, and combination treated tumors displayed significantly reduced ki-67 expression compared with control untreated tumors, and the difference was also statistically significant for the combination compared with cisplatin. Cleaved caspase-3 expression was significantly higher for sunitinib single agent and combination therapy compared with untreated controls, and for combination therapy compared with cisplatin alone. CD31 expression was diminished for both single agents and combination therapy compared with untreated tumors.ConclusionsSunitinib is preclinically active against urothelial carcinoma, and enhances the activity of cisplatin probably by targeting the stroma.     

Pyrrolidine dithiocarbamate exerts anti-proliferative and pro-apoptotic effects in renal cell carcinoma cell lines.

BACKGROUND: The activation of nuclear factor-kappaB (NF-kappaB) has been implicated in the development, progression and metastasis of renal cell carcinoma (RCC). This study investigates the effect of pyrrolidine dithiocarbamate (PDTC), a NF-kappaB inhibitor, on two metastatic human RCC cell lines, ACHN and SN12K1. METHODS: RCC cell lines and normal cells were exposed to 25 or 50 microM of PDTC. Apoptosis was measured by flow cytometry and TdT-mediated nick end labelling methods. Cell viability and proliferation were measured by MTT and BrdU assays, respectively. Expression of NF-kappaB subunits, IkappaBs, IkappaB Kinase (IKK) complex and apoptotic regulatory proteins were analysed by western blotting and/or immunofluorescence. DNA-binding activity of NF-kappaB subunits were measured by ELISA. RESULTS: RCC cell lines had a higher basal level expression of all the five subunits of NF-kappaB than normal primary cultures of human proximal tubular epithelial cells or HK-2 cells. PDTC decreased the viability and proliferation of RCC, but not normal cells. Of the two RCC cell lines, ACHN had a higher basal level expression of all the five NF-kappaB subunits than SN12K1 and was more resistant to PDTC. While PDTC induced an overall decrease in expression of all the five NF-kappaB subunits in both RCC cell lines, unexpectedly, it increased the nuclear expression of NF-kappaB in ACHN, but not in SN12K1. PDTC reduced the DNA-binding activity of all the NF-kappaB subunits and the expression of the IKK complex (IKK-alpha, IKK-beta and IKK-gamma) and the inhibitory units IkappaB-alpha and IkappaB-beta. PDTC induced a significant increase in apoptosis in both RCC cell lines. This was associated with a decrease in expression of the anti-apoptotic proteins, Bcl-2 and Bcl-(XL), without marked changes in the pro-apoptotic protein Bax. CONCLUSION: These data suggest that PDTC has the potential to be an anticancer agent in some forms of RCC.     

Inhibition of androgen-sensitive LNCaP prostate cancer growth in vivo by melatonin: association of antiproliferative action of the pineal hormone with mt1 receptor protein expression

BACKGROUND: Potential involvement of the mt1 receptor in the antiproliferative action of melatonin on androgen-sensitive LNCaP cells, and melatonin-induced modulation of androgen-insensitive PC-3 cell growth, have been reported in vitro. The effects of melatonin on prostate cancer cell proliferation and their association with mt1 receptor expression were investigated in athymic nude mice xenograft models of LNCaP and PC-3 cells. METHODS: Daily saline or melatonin (4 microg/g body weight) was given to nude mice before or after tumor cell inoculation. Tumor volume was measured periodically, and expression of PCNA, cyclin A, PSA, and mt1 receptor was assessed by immunohisto(cyto)chemistry and/or Western blotting. RESULTS: Melatonin inhibited the growth of LNCaP tumors, without affecting the growth of PC-3 xenografts, in nude mice. It induced significant decreases in the expression of PCNA, cyclin A, and PSA in LNCaP tumors. Expression of mt1 receptor protein was demonstrated in LNCaP cells, but not in PC-3 cells, both in vivo and in vitro. CONCLUSIONS: The antiproliferative action of melatonin on LNCaP tumor growth was demonstrated in vivo, and its association with mt1 receptor protein expression suggests the potential involvement of the receptor in the antitumor activity of the pineal gland hormone.     

StealthTMRNAi沉默人肝癌细胞Bcl-2表达抑制体外移植瘤生长的研究

目的探讨StealthTMRNAi抑制Bcl-2基因表达对体内移植瘤生长的影响。方法通过合成的人肝癌Bcl-2基因干扰序列转染入肝癌细胞内并从皮下移植到裸鼠，免疫组化检测瘤组织中Bcl-2蛋白的表达。结果荧光显微镜下显清晰的绿色荧光为转染成功细胞；RT．PCR检测转染组Bcl-2基因mRNA的表达水平下调（P〈0．01）。转染StealthTMRNA裸鼠皮下移植肿瘤生长缓慢，与对照组瘤体积比较差异有显著性（P〈0．01）；采用免疫组化法检测注射StealthTMRNA组，Bcl-2蛋白的表达下调（P〈0．01）。结论StealthTMRNAi可抑制Bcl-2基因表达，对移植瘤的生长有抑制作用。     

ACHN人肾癌裸鼠移植瘤模型的生物学特性及放射治疗观察

目的观察ACHN人肾癌裸鼠皮下移植瘤模型的生物学特性及其对放射线的敏感性。方法分别利用瘤细胞悬液接种法、瘤块包埋法获得ACHN人肾癌裸鼠移植瘤模型，观察移植瘤的生长情况、成瘤率、肝肺转移率及病理形态。ACHN皮下荷瘤鼠14只，随机分为空白对照组和放疗组，6MV X直线加速器对放疗组移植瘤局部单次照射12Gy，观察裸鼠的耐受性及肿瘤生长抑制情况；透射电镜观察瘤细胞超微结构变化。结果瘤块包埋法成瘤稳定，成瘤率92．5%，肝转移率43％，未见肺转移。裸鼠对12Gy射线局部照射耐受良好，放疗组移植瘤与空白对照组比较生长明显受抑制（P〈0．01）；放疗组瘤细胞凋亡现象明显。结论ACHN人肾癌裸鼠移植瘤模型可作为肾癌放疗实验研究的有效工具。