【背景】前列腺癌（prostate cancer，PCa）是男性生殖系最常见的恶性肿瘤，其发病率和死亡率逐年升高^[1]。晚期前列腺癌发生转移扩散不能手术根治，而雄激素剥夺治疗短期内有效，长期会引起去势抵抗性前列腺癌^[2]。所以需要研究其侵袭、转移和去势抵抗的分子机制，找出新的治疗靶点。最新研究表明缺氧是许多实体肿瘤微环境改变的主要特征，局部缺氧时肿瘤恶性程度加重^[3] 。本课题组前期研究发现槲皮素（quercetin ，Que）与2-甲氧雌二醇（2-methoxyestradiol ，2-ME）联用起到协同作用，显著抑制前列腺癌细胞恶性生长、缩小荷瘤小鼠移植瘤体积，且达到相同抑制效果时两者联用所需药物剂量较单独给药明显减少^[4]。因此，本文以肿瘤缺氧微环境为着眼点，继续研究两药联用是否协同抑制缺氧条件下前列腺癌细胞的恶性生长，为深入了解前列腺癌晚期侵袭转移的分子机制和化疗方案提供思路，也为Que联合2-ME的临床应用提供理论基础。

【目的】本文内容分为三部分。第一部分主要研究缺氧微环境对人前列腺癌细胞恶性生长的影响及关键信号转导分子表达变化；第二部分主要研究缺氧微环境下Que和2-ME联用对前列腺癌的抑制效果；第三部分主要研究两药联用抗癌的作用机制。

【方法】选用雄激素依赖性前列腺癌细胞LNCaP和雄激素非依赖性前列腺癌细胞PC3，缺氧（1%O2、5%CO2和94%N2）条件下培养^[5]。使用CCK-8法检测细胞增殖活性，计算Que（0、7.5、15、30、60、120、240 µM）和2-ME（0、0.25、0.5、2、4、8、16 µM）不同浓度处理后细胞生长抑制率，绘制量效曲线，计算IC50。根据拟合的量效曲线和IC50值，分别选择Que和2-ME的4个作用浓度，按照完全随机分组两因素析因设计，构成16个两药联用配伍组合并分别测定其对癌细胞的生长抑制率。根据Chou和Talalay的药物联用方程式计算药物联用指数（combination index，CI）^[6]。采用qRT-PCR和Western t方法检测缺氧诱导因子1α（hypoxia inducible factor -1α，HIF-1α）、上皮间充质转化（epithelial-mesenchyme transformation ，EMT）中标志分子mRNA和蛋白表达水平的变化。通过细胞划痕实验和Transwell实验检测癌细胞的转移和侵袭能力。采用小干扰RNA （small interfering RNA，siRNA）干扰技术降低HIF-1α的表达。 

【结果】第一部分结果显示，短时间缺氧状态下PC3和LNCaP细胞增殖活性均显著增强，分别在4 h和16 h最高；但长时间缺氧后，两种细胞增殖活性均下降，分别在20 h和72 h时最低。在不同缺氧阶段，与常氧对照组比较，两种前列腺癌细胞的迁移和侵袭能力均显著增高; HIF-1α表达增高并随缺氧时间延长呈现先增加后降低的趋势；同时，各缺氧组E-cadherin表达显著降低，而Vimentin表达显著增高。

第二部分结果显示，缺氧微环境下随Que或2-ME作用浓度增高，LNCaP或PC3细胞生长抑制率逐渐增加。对LNCaP细胞，Que和2-ME的IC50值分别为39.95 µM和3.34 µM。对PC3细胞，Que和2-ME的IC50值分别为40.48 µM 和4.35 µM。选取Que（10、20、40、60 µM）和2-ME（0.5、2、4、6 µM）组成16个配伍组判断药物联用效果。对LNCaP细胞，低剂量Que（10和20 µM）联合低剂量2-ME（0.5µM）及Que+2-ME（20+2µM）表现出协同作用（CI值<0.9）。而对PC3 细胞，除了低剂量Que（10 µM）联合低剂量2-ME（0.5和2 µM）及Que+2-ME（20+2µM）配伍组外，高剂量Que（60 µM）联合高剂量2-ME（4 µM）也表现出协同作用（CI值<0.9）。选取Que+2-ME（20+2µM）组合进行药物联用机制研究。

第三部分结果：首先观察缺氧条件下Que和2-ME 联用对关键信号转导分子表达的影响。与缺氧对照组比较，药物联用组HIF-1α、Vimentin分子表达明显降低而E-cadherin表达显著升高。其次，采用siRNA干扰降低HIF-1α表达进一步观察它在药物联用抗癌中的作用。结果显示LNCaP和PC3分别采用siRNA-1和siRNA-3干扰效率最高，达75%以上。与对照组比较，干扰组癌细胞增殖活性、迁移率和侵袭能力、Vimentin表达均显著降低；而E-cadherin表达增高。与药物联用组比较，干扰给药组细胞增殖活性、迁移率和侵袭能力、HIF-1α的表达均显著降低；同时E-cadherin分子表达明显升高； 而Vimentin的表达仅在LNCaP细胞中干扰给药组比药物联用组显著降低，PC3细胞中表达无差异。

结论：缺氧促进前列腺癌的恶性生长是一个动态变化的过程。Que与2-ME联合用药协同抑制前列腺癌细胞恶性进展并呈剂量相关性，其作用机制可能与HIF-1α介导的EMT逆向转化有关。

Background:  Prostate cancer (PCa) is the most common malignant tumor of the male genital system, and its morbidity and mortality rates are increasing year by year . Advanced prostate cancer that has metastasized and spread cannot be cured surgically. While androgen deprivation therapy is effective in the short term, but causes desmoid-resistant prostate cancer in the long term. Therefore, there is a need to study the molecular mechanisms of its invasion, metastasis and denudation resistance and to explore new therapeutic targets. Recent studies have shown that hypoxia is a major feature of the altered microenvironment of many solid tumors, and that  the malignant degree of tumors is aggravated when local hypoxia occurred, such as the ability to proliferate, migrate, and invade. Our group found that the combination of quercetin (Que) and 2-methoxyestradiol (2-ME) had a synergistic effect, significantly inhibiting the malignant growth of prostate cancer cells and reducing the size of transplanted tumors in tumor-bearing mice, and the drug dose required for the same inhibitory effect was significantly reduced compared with that of the drug alone. Therefore, this paper focuses on the tumor hypoxic microenvironment and continues to investigate whether the combination of the two drugs synergistically inhibits the malignant growth of prostate cancer cells under hypoxic conditions, providing ideas for an in-depth understanding of the molecular mechanisms and chemotherapeutic regimens for advanced invasive metastasis of prostate cancer, as well as providing a theoretical basis for the clinical application of Que combined with 2-ME.

Objective:  This paper is divided into three main parts. The first part focuses on the effect of hypoxic microenvironment on the malignant growth of human prostate cancer cells and the changes in the expression of key signal transduction molecules; the second part focuses on the inhibitory effect of Que and 2-ME combination on prostate cancer under hypoxic microenvironment; the third part focuses on the mechanism of action of the combination of the two drugs against cancer.

Methods:  The androgen-dependent prostate cancer cell line LNCaP and the androgen-independent prostate cancer cell line PC3 were selected and cultured under hypoxic conditions (1% O₂, 5% CO₂ and 94% N₂) . The cell proliferation activity was measured by CCK-8 method, and the cell growth inhibition rate was calculated after treatment with different concentrations of Que (0, 7.5, 15, 30, 60, 120, 240 µM) and 2-ME (0, 0.25, 0.5, 2, 4, 8, 16 µM), and the quantitative efficacy curve was plotted to calculate the half maximal inhibitory concentration (IC50). Based on the fitted dose-effect curves and IC50 values, four concentrations of Que and 2-ME were selected to form 16 combinations of the two drugs and their growth inhibition rates were determined according to a two-factor analysis design with completely randomized groups. Calculation of drug combination index (CI) based on Chou and Talalay's drug combination equation. qRT-PCR and Western Blot were used to detect the changes in mRNA and protein expression levels of the molecular biomarkers in hypoxia inducible factor 1α (HIF-1α), epithelial-mesenchyme transformation (EMT) and protein expression levels in EMT. The metastatic and invasive ability of cancer cells was detected by cell scratch assay and Transwell assay. The expression of HIF-1α was reduced by small interfering RNA (siRNA) interference technology.

Results: 

PART I RESULTS: The results showed that the proliferation activity of both PC3 and LNCaP cells was significantly enhanced under short time hypoxia, and was highest at 4 h and 16 h, respectively, compared with other time groups; however, the proliferation activity of both cells decreased after prolonged hypoxia, and reached the lowest at 20 h and 72 h, respectively (P<0.05). The migration rate and invasive ability of both prostate cancer cells were significantly higher in different hypoxic stages compared with the normoxic group (P<0.01). The mRNA and protein expression levels of HIF-1α were significantly higher in the hypoxic groups than in the normoxic groups, and showed an overall trend of increasing and then decreasing with the prolongation of hypoxia (P<0.05); the mRNA and protein expression levels of E-cadherin were significantly lower in the hypoxic groups than in the normoxic groups (P<0.05); the mRNA and protein expression levels of Vimentin were significantly higher in the hypoxic groups than in the normoxic groups (P<0.05). The mRNA and protein expression levels of Vimentin in all hypoxic groups were significantly higher than those in the normoxic group (P<0.05).

PART II RESULTS: the results showed that the growth inhibition rate of LNCaP or PC3 cells gradually increased with increasing concentrations of Que or 2-ME in the hypoxic microenvironment. The IC50 values of Que and 2-ME were 39.95 µM and 3.34 µM for LNCaP cells, respectively, and 40.48 µM and 4.35 µM for PC3 cells, respectively. Que (10, 20, 40, 60 µM) and 2-ME (0.5, 2, 4, 6 µM) were selected according to the dose-effect curves and IC50 values to determine the drug combination effects in 16 randomized dosing groups. For LNCaP cells, low doses of Que (20 µM) combined with low doses of 2-ME (0.5 and 2 µM) and Que+2-ME (20+2 µM) showed synergistic effects (CI <0.9). In contrast, for PC3 cells, the high dose of Que (60 µM) combined with the high dose of 2-ME (4 µM) also showed a synergistic effect (CI < 0.9), in addition to the low dose of Que (10 µM) combined with the low dose of 2-ME (0.5 and 2 µM) and Que+2-ME (20+2 µM) pairing groups. Que+2-ME (20+2 µM) was selected as the best pairing combination for subsequent mechanistic studies of drug combinations.

PART III RESULTS: Firstly, we observed the effect of Que and 2-ME combination on the expression of key signal transduction molecules in cancer cells under hypoxic conditions. The mRNA and protein expression of HIF-1α were significantly lower in the drug combination group compared with the drug alone group (P<0.05). Meanwhile, the mRNA and protein expression of E-cadherin were significantly higher in the drug combination group compared with the drug alone group (P<0.05). In contrast, mRNA and protein expression of Vimentin were significantly lower in the drug combination group compared with the drug alone group (P<0.05). Secondly, siRNA interference was used to reduce HIF-1α expression to observe whether HIF-1α was a key target of drug combination against cancer. The results showed that LNCaP and PC3 were interfered with siRNA-1 and siRNA-3 respectively with the highest efficiency of more than 75% (P<0.05). The cell proliferation activity, migration rate and invasion ability of the interfered group were significantly lower than those of the control group (P<0.05). Meanwhile, the mRNA and protein expression of E-cadherin were significantly higher in the interference group than in the control group (P<0.05), while the mRNA and protein expression of Vimentin were significantly lower than in the control group (P<0.05). Compared with the drug combination group, the cell proliferation activity, migration rate and invasion ability were significantly lower in the interference administration group (P<0.05); the expression of HIF-1α was significantly lower in the interference administration group than in the drug combination group (P<0.05); meanwhile, the protein expression of E-cadherin was significantly higher in the interference administration group than in the drug combination group for LNCaP cells (P<0.05), while the mRNA expression was not significantly different in the interference administration group than in the drug combination group for PC3 cells (P<0.05). The mRNA expression was not significantly different, while for PC3 cells, the expression of E-cadherin was significantly higher (P<0.05) in the interference administration group compared with the drug combination group; for LNCaP cells, the expression of Vimentin was significantly lower (P<0.05) in the interference administration group compared with the drug combination group, while for PC3 cells, the expression of Vimentin was significantly lower (P<0.05) in the interference administration group compared with the drug combination group. The expression of Vimentin in PC3 cells was not significantly different from that in the drug combination group.

Conclusion: Hypoxia promotes malignant growth of prostate cancer is a dynamic process. Que synergistically inhibits malignant progression of prostate cancer cells in combination with 2-ME in a dose-related manner. The mechanism of action may be related to HIF-1α-mediated reverse transformation of EMT.