Gan Y, Wientjes M G, Schuller D E, Au J L
Division of Pharmaceutical Chemistry, College of Pharmacy, Ohio State University, Columbus, Ohio 43210, USA.
Cancer Res. 1996 May 1;56(9):2086-93.
The pharmacodynamics of taxol in human head and neck squamous cell carcinoma were studied using histocultures of surgical specimens from patients (n = 22). Tumors were treated with taxol for 24 h. The inhibition of DNA synthesis was determined by 48 h cumulative bromodexyuridine (BrdUrd) incorporation. The induction of apoptosis was measured by morphological changes, in situ DNA end labeling, post-exonuclease III BrdUrd labeling, and DNA fragmentation. Inhibition of the BrdUrd labeling index (LI) by taxol was incomplete, with 11 tumors showing maximal inhibition (Emax) of 30-50% and the remaining 11 tumors showing and Emax of 50-80%. For both groups, the inhibition approached maximum values at 1 microM taxol concentration; an additional 10-fold increase in drug concentrations did not significantly enhance the inhibition. The taxol concentrations required for a 30% inhibition (IC30) were 4.2 and 0.3 microM for the first and second groups, respectively. The IC30 correlated with the Emax (r2 = 0.39; P < 0.001). Taxol induced apoptosis in all tumors, 11 tumors showed a maximal fraction of apoptotic tumor cells between 3 and 10% and 11 tumors between 13 and 28%, whereas untreated controls showed a maximal apoptotic index of < 1%. For individual tumors, the maximal apoptotic index occurred between 0.1 and 3 microM, and correlated with the BrdUrd LI for the untreated control (r2 = 0.37; P < 0.01). It is interesting that > 95% of apoptotic cells were BrdUrd labeled, whereas not all BrdUrd-labeled cells were apoptotic. To investigate the basis of the variable tumor response to taxol, we determined the expression of multidrug resistance P-glycoprotein (Pgp), p53, and bcl-2 proteins, using immunohistochemical staining and Western blot analysis. Eleven (50%), 10 (45%), and 7 (32%) tumors expressed Pgp, p53, and bcl-2, respectively. Patients with Pgp-positive tumors showed a higher number of affected lymph nodes than those with Pgp-negative tumors (P < 0.05). Compared with moderately and well differentiated tumors, the poorly differentiated tumors expressed p53 and Pgp more frequently and showed a lower maximum inhibition of DNA synthesis and a higher apoptotic fraction after taxol treatment (P < 0.05 in both cases). Pgp expression correlated differently with taxol-induced inhibition of DNA synthesis than with apoptosis; Pgp-positive tumors showed a significantly higher Emax (63%) and IC30 (4.2 microM) but also a higher apoptotic index (17%) than Pgp-negative tumors (Emax 36%; IC30, 0.3 microM; and apoptotic index; 6%; P < 0.05 for all cases). p53 and bcl-2 expression did not correlated with taxol-induced inhibition of DNA synthesis or apoptosis. The data indicate that taxol acts through apoptosis and inhibition of proliferation in human head and neck cancer. Pgp overexpression appears to protect cells from the antiproliferative effect of taxol but correlated with a higher apoptosis.
利用22例患者手术标本的组织培养研究了紫杉醇在人头颈部鳞状细胞癌中的药效学。肿瘤用紫杉醇处理24小时。通过48小时累积溴脱氧尿苷（BrdUrd）掺入来测定DNA合成的抑制。通过形态学变化、原位DNA末端标记、核酸外切酶III后BrdUrd标记和DNA片段化来测量细胞凋亡的诱导。紫杉醇对BrdUrd标记指数（LI）的抑制不完全，11个肿瘤显示最大抑制（Emax）为30 - 50%，其余11个肿瘤显示Emax为50 - 80%。对于两组，在紫杉醇浓度为1μM时抑制接近最大值；药物浓度再增加10倍并没有显著增强抑制作用。第一组和第二组达到30%抑制（IC30）所需的紫杉醇浓度分别为4.2μM和0.3μM。IC30与Emax相关（r2 = 0.39；P < 0.001）。紫杉醇在所有肿瘤中诱导细胞凋亡，11个肿瘤显示凋亡肿瘤细胞的最大比例在3%至10%之间，11个肿瘤在13%至28%之间，而未处理的对照显示最大凋亡指数<1%。对于单个肿瘤，最大凋亡指数出现在0.1至3μM之间，并且与未处理对照的BrdUrd LI相关（r2 = 0.37；P < 0.01）。有趣的是，>95%的凋亡细胞被BrdUrd标记，而并非所有被BrdUrd标记的细胞都是凋亡细胞。为了研究肿瘤对紫杉醇反应不同的基础，我们使用免疫组织化学染色和蛋白质印迹分析来测定多药耐药P - 糖蛋白（Pgp）、p53和bcl - 2蛋白的表达。分别有11个（50%）、10个（45%）和7个（32%）肿瘤表达Pgp、p53和bcl - 2。Pgp阳性肿瘤的患者比Pgp阴性肿瘤的患者有更多的受累淋巴结（P < 0.05）。与中分化和高分化肿瘤相比，低分化肿瘤更频繁地表达p53和Pgp，并且在紫杉醇处理后显示出较低的DNA合成最大抑制和较高的凋亡分数（两种情况均P < 0.05）。Pgp表达与紫杉醇诱导的DNA合成抑制和细胞凋亡的相关性不同；Pgp阳性肿瘤显示出显著更高的Emax（63%）和IC30（4.2μM），但也比Pgp阴性肿瘤有更高的凋亡指数（17%）（Emax 36%；IC30，0.3μM；凋亡指数；6%；所有情况P < 0.05）。p53和bcl - 2表达与紫杉醇诱导的DNA合成抑制或细胞凋亡无关。数据表明紫杉醇通过细胞凋亡和抑制人头颈部癌的增殖起作用。Pgp的过度表达似乎保护细胞免受紫杉醇的抗增殖作用，但与更高的细胞凋亡相关。
