表阿霉素和阿霉素对原代乳腺癌的体外敏感性研究

目的 了解表阿霉素和阿霉素在体外对乳腺癌、癌旁组织和正常乳腺组织的杀伤作用。方法 取得不同患者的乳腺癌、癌旁和正常乳腺组织标本，采用胶原酶消化法获取乳腺原代细胞，应用原代体外培养和MTT法检测化疗药物在不同浓度下对乳腺原代细胞的杀伤率。结果 在1倍PPC（血药浓度）时表阿霉素对乳腺癌原代细胞的体外肿瘤杀伤作用强于阿霉素（P〈0．01）；对正常的乳腺原代细胞，表阿霉素和阿霉素的体外杀伤作用明显下降（P〈0．01），而且表阿霉素弱于阿霉素（P〈0．01）。结论 对于原发乳腺癌患者，表阿霉素不但治疗效果优于阿霉素，而且对肿瘤组织具有更高的选择性，可以作为乳腺癌治疗的一线药物。     

原发和复发乳腺癌体外化疗药物敏感性的量效关系研究

目的：通过体外研究探讨化疗剂量对乳腺癌治疗效果的影响。方法：通过手术取材和肿瘤组织原代细胞体外培养技术，对96例乳腺癌患者进行体外药物敏感性检测。结果：乳腺癌晚期患者比早期患者的耐药风险明显增加（P〈0．05），乳腺癌复发患者比原发患者具有更强的多药耐药能力（P〈0．01）；增加化疗药物剂量仍然可以提高大多数耐药患者的治疗效果（P〈0．01）。结论：大剂量化疗方案对晚期和复发性乳腺癌患者有帮助。     

MTT法测定复发转移性乳腺癌体外化疗药物敏感性实验研究

目的探讨MTT法体外药敏试验在复发转移性乳腺癌化疗中的价值。方法取35例复发转移性乳腺癌患者肿瘤组织，简易机械法制备细胞悬液，应用MTT法体外药敏试验测定其对10种常用化疗药物的敏感性，并指导临床用药，同时与临床疗效进行相关分析。结果MTT法检测复发转移乳腺癌患者药敏试验的成功率为91．4％，体外药敏试验的敏感性为86．4％，特异性为80．0％，阳性预测值为90．5％，阴性预测值为72．7％，总的预测准确率为84．4％。结论MTT法可作为临床上指导复发转移乳腺癌个体化化疗的有效方法。     

利用体外药敏试验指导膀胱癌临床个体化灌注化疗

目的研究根据体外药敏试验结果选择药物来指导临床膀胱癌患者的个体化膀胱内灌注化疗。方法选取25例非肌层浸润性膀胱癌患者手术标本作原代细胞培养及化疗药物敏感试验,选择敏感的化疗药物对患者进行膀胱内灌注治疗,随访7年观察肿瘤复发率。结果25例组织标本中24例完成实验。各化疗药物总体敏感率差异无统计学意义（P〉0．05）,但每种化疗药物对不同个体的敏感率差异较大。24例患者膀胱灌注化疗的5年复发率为16．7%（4／24）,7年复发率为25．0％（6／24）,5例对4种化疗药物均耐药的患者中3例复发。结论体外药敏试验筛选的药物与膀胱内灌注化疗临床疗效间有良好的相关性,为个体化治疗提供了有价值的临床指导。     

原代培养的肺癌细胞对TIL的反杀伤作用

目的：探讨肺癌细胞对TIL细胞是否具有反杀伤作用及意义。方法：用MTF法检测8例人原发性肺癌的原代培养肿瘤细胞被Fas配体（Fas ligand,FasL）抗体封闭前后对肿瘤浸润淋巴细胞（Tumor Infiltrating Lymphocytes。TIL）的杀伤活性。结果：发现原代培养的肺癌细胞被FasL抗体封闭前后对TIL均有不同程度的杀伤作用．但经FasL抗体封闭后的肿瘤细胞对TIL的杀伤活性均有所下降。两组之间存在显著差异（P〈0．05）。结论：提示肺癌细胞对TIL细胞的反杀伤作用可能是其逃避免疫攻击的机制之一。     

热化疗诱导胶质瘤细胞凋亡的实验研究

目的 研究热化疗对C6细胞凋亡的影响，评价热化疗抑制细胞增殖的价值。方法 将不同浓度的丝裂霉素直接作用于体外培养的C6胶质瘤细胞，接种后20h,48h接种后42．7℃恒温箱中加温2次，每次30min。观察丝裂霉素对C6细胞的抑制率，生长曲线的影响及热疗，化疗，热化疗对C6细胞凋亡的诱导作用。结果 丝裂霉素在1.5μg/ml浓度时杀伤作用达高峰（P＜0．001），再加大浓度并不增加杀伤作用（P＞0．05）；透射电镜，FCM，TUNEL均观察到热疗，化疗，热化疗诱导细胞凋亡的作用（P＜0．001），热化疗有协同作用（P＜0．001）。结论 （1）热疗，化疗，热化疗均抑制C6细胞增殖。（2）热疗，化疗，热化疗诱导细胞凋亡为其抗癌机制之一。（3）热疗能提高化疗药物的细胞毒性作用。     

雌二醇对不同年龄阶段患者的乳腺癌原代细胞增殖影响的体外研究

目的:了解在体外条件下不同浓度的雌二醇对不同年龄阶段患者的乳腺癌原代细胞增殖的影响。方法:用胶原酶消化法获取不同年龄阶段患者的乳腺癌原代细胞,并用MTT法检测乳腺癌原代细胞在体外培养下,给予不同浓度雌二醇时,细胞的生长增殖情况。结果:雌二醇对所有ER表达阳性的乳腺癌原代细胞均有刺激生长的作用,而且作用基本相同;对所有ER表达阴性的乳腺癌原代细胞,雌激素并没有明显的刺激生长作用。结论:当体内的雌二醇水平≥103pmol/L时,乳腺癌的发病风险和复发风险会显著增加,并且与患者的卵巢功能无关。     

LDH释放法检测肿瘤患者PBMC和CIK细胞的细胞毒活性

目的建立检测细胞毒活性的乳酸脱氢酶（LDH）测定法，了解肿瘤患者外周血单个核细胞（PBMC）和细胞因子诱导的杀伤细胞（CIK）的抗肿瘤活性，为肿瘤的免疫细胞治疗提供参考。方法采集肿瘤患者和健康成人外周血，常规分离PBMC，体外进行CIK的培养，用LDH释放法分别检测PBMC和CIK对肿瘤细胞K562的细胞毒活性。结果肿瘤患者PBMC对K562的杀伤活力比正常人显著降低（P〈0．05）。经多种细胞因子诱导培养7～10d后，健康人和肿瘤患者的CIK对K562的杀伤活力都有显著提高（P〈0．01），肿瘤患者CIK杀伤活力接近于健康人。结论肿瘤患者PBMC细胞毒活性显著降低，但经诱导培养成CIK后则显著提高。用LDH释放法检测肿瘤病人CIK细胞毒活力，有助于CIK疗法适应病人的选择及个体疗效观察。     

乳腺癌类器官疾病模型的构建及其对化疗药物的敏感性试验

目的 建立及鉴定患者来源的乳腺癌类器官模型，并利用该模型进行个体化的化疗药物敏感性检测。方法 从在医院接受乳腺癌根治术的4例女性患者手术标本（不同乳腺癌分子亚型）获取部分肿瘤组织处理后获取乳腺癌细胞，基质胶重悬乳腺癌细胞并接种于培养皿中进行三维培养；制备石蜡切片并进行形态学和免疫组织化学（ER、PR、HER-2和Ki-67）染色，通过与亲本肿瘤组织对比，检测其能否还原体内肿瘤的组织病理学特征；利用不同浓度的7种化疗药物处理乳腺癌类器官5 d后，使用CellTiterGlo?3D试剂测定细胞活力，分析药敏结果。结果 乳腺癌类器官与其来源的患者肿瘤在组织病理学特征上高度一致。不同分子亚型乳腺癌类器官对化疗药物的敏感性不同。结论 本研究成功构建的乳腺癌类器官模型能够反映亲本乳腺肿瘤的组织学分型，并能够进行体外化疗药物敏感性试验，有望为患者临床用药提供参考。     

肿瘤细胞裂解物致敏树突状细胞对小鼠乳腺癌作用的研究

目的观察肿瘤细胞裂解物致敏树突状细胞 (DC)对小鼠乳腺癌的治疗作用。方法无菌取小鼠骨髓细胞 ,在体外培养条件下经细胞因子作用诱导为树突状细胞 (DC) ,用EMT6肿瘤抗原裂解物冲击致敏DC细胞 ,检测DC体外刺激活化淋巴细胞作用 ,以及经DC免疫产生的细胞毒T淋巴细胞 (CTL)体外杀伤肿瘤细胞的活性 ,观察致敏DC免疫对小鼠乳腺癌模型的治疗作用。结果镜下可见抗原致敏后的DC可吸引淋巴细胞聚集成团 ;致敏DC诱导生成的特异性CTL在体外对肿瘤细胞可产生杀伤作用 (与PBS对照组比较 ,P =0 .0 2 7) ,而未成熟DC细胞组和肿瘤抗原组与PBS对照组间无显著性差异 (P =0 .17,P =0 .0 72 ) ;经致敏DC注射免疫后 ,小鼠负荷肿瘤得到抑制 (与PBS对照组比较 ,P =0 .0 3 5 ) ,而单纯肿瘤抗原及未致敏DC免疫组与PBS对照组间无显著性差异 (P =0 .2 6,P =0 .11)。结论肿瘤抗原裂解物致敏的DC可有效递呈抗原并诱导淋巴细胞杀伤肿瘤细胞。     

聚乳酸-羟基乙酸共聚物微球瘤内注射抑制荷人三阴性乳腺癌裸鼠移植瘤细胞的增殖

背景:通过超声引导,将抗肿瘤药物缓释剂注射到肿瘤局部进行间质化疗,可提高抗肿瘤效果,并且减轻全身毒副作用。目的:观察超声引导下瘤内注射多西他赛聚乳酸-羟基乙酸微球对荷人三阴性乳腺癌裸鼠移植瘤增殖的抑制作用。方法:采用乳化溶剂挥发法制备多西他赛聚乳酸-羟基乙酸微球,扫描电镜观察微球的表面形态及粒径,高效液相色谱法测定包封率、载药率及体外释放情况。建立荷人三阴性乳腺癌裸鼠移植瘤模型,随机分为5组:模型组无处理;微球组在肿瘤内注射空白聚乳酸-羟基乙酸微球1次;多西他赛瘤内注射组在肿瘤内注射多西他赛注射液(多西他赛剂量为10mg/kg),每10d给药1次,连续4次;多西他赛聚乳酸-羟基乙酸共聚物微球低剂量组在肿瘤内注射载药微球(多西他赛剂量为20mg/kg)1次;多西他赛聚乳酸-羟基乙酸共聚物微球高剂量组在肿瘤内注射载药微球(多西他赛剂量为40mg/kg)1次。结果与结论:多西他赛聚乳酸-羟基乙酸微球平均粒径23.1μm,包封率为96.3%,载药率为4.82%,40d累积释放药物85.7%。超声引导下多西他赛聚乳酸-羟基乙酸微球瘤体内注射具有明显的抗肿瘤作用,高剂量组抑瘤率达65.7%,彩色多普勒超声见肿瘤血流信号明显减少,病理学检查见肿瘤组织大片坏死,提示超声引导下瘤内注射多西他赛聚乳酸-羟基乙酸微球可明显抑制荷人三阴性乳腺癌裸鼠移植瘤增殖。     

Chemotherapy dosing schedule influences drug resistance development in ovarian cancer

Drug resistance leads to chemotherapy failure and is responsible for the death of a great majority of patients with metastatic, late-stage ovarian cancer. The present study addressed whether changes in the chemotherapy dosing schedule affect the development, further worsening, or circumvention of drug resistance in chemosensitive and chemoresistant ovarian cancer. Severe combined immunodeficient mice bearing HeyA8 and HeyA8-MDR xenografts were treated with docetaxel intermittently (1×/wk or 3×/wk) or continuously for 21 days. Tumor mRNA expression of genes implicated in docetaxel resistance was measured by quantitative real-time-PCR. Analyzed genes included those encoding for the drug efflux transporters mdr1 and mrp7 and for molecules that interfere with or overcome the effects of docetaxel, including β-tubulinIII, actinin4, stathmin1, bcl2, rpn2, thoredoxin, and akt2. In both models, continuous docetaxel resulted in greater antitumor efficacy than 1×/wk or 3×/wk dosing and did not induce upregulation of any analyzed genes. Once weekly dosing caused upregulation of various drug resistance-related genes, especially in chemoresistant xenografts. More frequent, 3×/wk dosing diminished this effect, although levels of various genes were higher than for continuous chemotherapy. Drug efflux transporter expression was further examined by Western blotting, confirming that intermittent, but not continuous, docetaxel induced significant upregulation. Overall, our results show that the presence and length of treatment-free intervals contribute to the development of drug resistance. Elimination of these intervals by continuous dosing resulted in superior antitumor efficacy and prevented drug resistance induction in chemosensitive and chemoresistant disease. These results encourage the clinical implementation of continuous chemotherapy to overcome and/or prevent drug resistance in newly diagnosed and recurrent, refractory ovarian cancer.     

The mechanism of methylselenocysteine and docetaxel synergistic activity in prostate cancer cells

The study was designed to evaluate the combination treatment of methylselenocysteine (MSeC) and docetaxel and to delineate the underlying mechanism associated with observed in vitro synergy between MSeC and docetaxel in prostate cancer cells. Cells were treated with different concentrations and schedules (concurrent or sequential) of MSeC and docetaxel alone or in combination. Cell growth/death was assessed with sulforhodamine B assay, trypan blue assay, and time-lapse video. Loewe synergism/antagonism model was used to determine whether the combination effect was additive, synergistic, or antagonistic. Apoptosis and caspase-3 activity were evaluated with cell death ELISA assay and caspase activity assay, respectively. Synergy between MSeC and docetaxel was further assessed in the presence and absence of z-VAD-fmk, a pan-caspase inhibitor. Effect of MSeC and docetaxel alone or in combination on the cellular expression of the antiapoptotic protein survivin was measured with Western blot analyses. Pretreatment with MSeC was crucial to enhance docetaxel antitumor activity. The enhanced antitumor activity of the sequential combination treatment of MSeC and docetaxel (MSeC/docetaxel) was highly synergistic. Apoptosis increased after MSeC/docetaxel, compared with each drug alone or concurrent treatment. Pretreatment with z-VAD-fmk converted the synergy into antagonism, suggesting that the synergy is caspase-dependent apoptosis. The survivin level was down-regulated following MSeC/docetaxel treatment when compared with each drug alone. In conclusion, pretreatment with MSeC was essential to markedly sensitize cells to docetaxel. The synergy between MSeC and docetaxel in C2G prostate cancer cells is associated with increased level of caspase-dependent apoptosis and decreased level of survivin.     

Natural BH3 mimetic (-)-gossypol chemosensitizes human prostate cancer via Bcl-xL inhibition accompanied by increase of Puma and Noxa

Antiapoptotic members of the Bcl-2 family proteins are overexpressed in prostate cancer and are promising molecular targets for modulating chemoresistance of prostate cancer. (-)-Gossypol, a natural BH3 mimetic, is a small-molecule inhibitor of Bcl-2/Bcl-xL/Mcl-1 currently in phase II clinical trials as an adjuvant therapy for human prostate cancer. Our objective is to examine the chemosensitization potential of (-)-gossypol in prostate cancer and its molecular mechanisms of action. (-)-Gossypol inhibited cell growth and induced apoptosis through mitochondria pathway in human prostate cancer PC-3 cells and synergistically enhanced the antitumor activity of docetaxel both in vitro and in vivo in PC-3 xenograft model in nude mouse. (-)-Gossypol blocked the interactions of Bcl-xL with Bax or Bad in cancer cells by fluorescence resonance energy transfer assay and overcame the Bcl-xL protection of FL5.12 model cells on interleukin-3 withdrawal. Western blot and real-time PCR studies showed that a dose-dependent increase of the proapoptotic BH3-only proteins Noxa and Puma contributed to the cell death induced by (-)-gossypol and to the synergistic effects of (-)-gossypol and docetaxel. The small interfering RNA knockdown studies showed that Noxa and Puma are required in the (-)-gossypol-induced cell death. Taken together, these data suggest that (-)-gossypol exerts its antitumor activity through inhibition of the antiapoptotic protein Bcl-xL accompanied by an increase of proapoptotic Noxa and Puma. (-)-Gossypol significantly enhances the antitumor activity of chemotherapy in vitro and in vivo, representing a promising new regime for the treatment of human hormone-refractory prostate cancer with Bcl-2/Bcl-xL/Mcl-1 overexpression.     

Elimination of Progressive Mammary Cancer by Repeated Administrations of Chimeric Antigen Receptor-Modified T Cells

Continuous oncogenic processes that generate cancer require an on-going treatment approach to eliminate the transformed cells, and prevent their further development. Here, we studied the ability of T cells expressing a chimeric antibody-based receptor (CAR) to offer a therapeutic benefit for breast cancer induced by erbB-2. We tested CAR-modified T cells (T-bodies) specific to erbB-2 for their antitumor potential in a mouse model overexpressing a human erbB-2 transgene that develops mammary tumors. Comparing the antitumor reactivity of CAR-modified T cells under various therapeutic settings, either prophylactic, prior to tumor development, or therapeutically. We found that repeated administration of CAR-modified T cells is required to eliminate spontaneously developing mammary cancer. Systemic, as well as intratumoral administered CAR-modified T cells accumulated at tumor sites and eventually eliminated the malignant cells. Interestingly, within a few weeks after a single CAR T cells'' administration, and rejection of primary lesion, tumors usually relapsed both in treated mammary gland and at remote sites; however, repeated injections of CAR-modified T cells were able to control the secondary tumors. Since spontaneous tumors can arise repeatedly, especially in the case of syndromes characterized by specific susceptibility to cancer, multiple administrations of CAR-modified T cells can serve to control relapsing disease.     

Aflibercept exerts antivascular effects and enhances levels of anthracycline chemotherapy in vivo in human acute myeloid leukemia models

We examined whether potent vascular endothelial growth factor (VEGF) blockade mediated by aflibercept, a decoy VEGF receptor (VEGFR) 1/2 moiety with stronger affinity for VEGF than bevacizumab, resulted in antileukemia effects and enhanced the efficacy of systemic chemotherapy. The efficacy of aflibercept alone and in combination with doxorubicin was evaluated in human VEGF-expressing acute myeloid leukemia (AML) cell lines and primary cells xenotransplanted into immunodeficient mice. Aflibercept reduced primary VEGF/VEGFR-positive AML colony formation growth in vitro and inhibited AML xenograft growth up to 93% in association with antiangiogenic and antiproliferative effects, hypoxia, and VEGF sequestration in multiple models. High VEGF-A expression by AML cells promoted in vivo xenograft growth and aflibercept sensitivity. Aflibercept therapy slowed disease progression in two systemic human AML xenograft models and reduced peripheral leukemia disease in a primary relapsed AML model in NOD/SCID/IL2Rγnull mice. Combination aflibercept and doxorubicin enhanced antitumor effects in local xenograft models. Sequential aflibercept followed by doxorubicin resulted in progressive anthracycline accumulation in marrow and extramedullary AML sites and resulted in 2-fold higher drug levels 24 hours after administration. In contrast, tissues (tumor, plasma, marrow) treated with chemotherapy only showed progressive drug clearance over time. Combination aflibercept and doxorubicin also resulted in vascular narrowing, decreased vessel number, and perivascular apoptosis. These data suggest that inefficient drug delivery by leukemia-associated vasculature may mediate chemoresistance and support further clinical evaluation of combination aflibercept and anthracycline therapy in refractory/relapsed AML patients.     

泰索帝与阿霉素对甲状腺未分化癌细胞(TA-K)作用比较及对细胞周期的影响

目的比较泰索帝与阿霉素对甲状腺未分化癌细胞(TA-K)的增殖抑制作用,研究泰索帝对甲状腺未分化癌细胞作用的机制。方法应用MTT方法观察泰索帝与阿霉素对TA-K细胞的抑制率,应用流式细胞仪探讨泰索帝对TA-K细胞增殖周期的影响。结果泰索帝对TA-K细胞增殖抑制作用大于阿霉素,泰索帝主要是使TA-K细胞抑制于G2/M期,促进细胞凋亡。结论泰索帝对甲状腺未分化癌的抑制作用优于阿霉素,有望成为甲状腺未分化癌更为有效的化疗药物。     

Combined targeting of epidermal growth factor receptor and hedgehog signaling by gefitinib and cyclopamine cooperatively improves the cytotoxic effects of docetaxel on metastatic prostate cancer cells

The epidermal growth factor receptor (EGFR) and hedgehog cascades provide a critical role in prostate cancer progression and contribute to the resistance to clinical therapies and disease relapse. Therefore, we evaluated, for the first time, the antiproliferative and cytotoxic effects induced by a combination of selective inhibitors of EGFR tyrosine kinase and smoothened hedgehog signaling element, gefitinib and cyclopamine, with a current chemotherapeutic drug used in the clinics, docetaxel, on some metastatic prostate cancer cell lines. Immunohistochemical analyses revealed that sonic hedgehog (SHH) expression was enhanced in 39% of primary prostatic adenocarcinomas (Gleason scores 4-10) compared with the corresponding normal tissues of the same prostate gland from 32 prostate cancer patients. The confocal microscopy and Western blot analyses have also indicated the high expression levels of SHH and EGFR in metastatic LNCaP, DU145, and PC3 cells. Moreover, the results revealed that the drugs, alone or in combination, at lower concentrations inhibited the growth of EGF plus SHH-stimulated and serum-stimulated androgen-responsive LNCaP-C33 and androgen-independent LNCaP-C81, DU145, and PC3 cells. Importantly, the combined docetaxel, gefitinib, and cyclopamine also caused a higher rate of apoptotic death of prostate cancer cells compared with individual agents. The cytotoxic effects induced by these drugs in PC3 cells seem to be mediated in part through the cellular ceramide production and activation of caspase cascades via a mitochondrial pathway and the release of cytochrome c into the cytosol. Additionally, the combined agents were more effective at suppressing the invasiveness of PC3 cells through Matrigel in vitro than the single drugs. These findings indicate that the combined use of inhibitors of EGF-EGFR and hedgehog signaling with docetaxel could represent a more promising strategy for treatment in patients with metastatic and androgen-independent prostate cancer.