金雀异黄素抗肝癌细胞的研究/

2019-05-11 06:54:06

mL 细胞 凋亡 genistein 金雀异



原发性肝癌(hepatocelluar carcinoma,HCC)是我国常见的恶性肿瘤之一,死亡率较高,年死亡率14.52/10万人口。此病常合并肝硬化,并早期即在肝内扩散,由于缺乏特异性症状,难以早期发现,加之病情进展迅速,大多数已失去了手术机会,而且95%的病人对放疗不敏感,全身化疗的疗效低,近期疗效仅在15%以下,故常于发病后短期内死亡,所以寻找毒副作用轻,又可抑制肿瘤细胞生长,促进凋亡的非细胞毒性药物,成为研究的重要方向。Hino等研究证实细胞凋亡存在于肝癌发展的各个阶段,并随着肿瘤的发展、其增殖率高于凋亡率。细胞凋亡是基因控制的细胞自主性死亡过程,大量研究证实,细胞凋亡与肝癌的发生、发展有密切的关系。因此,如何提高诱导肿瘤细胞凋亡,同时降低毒副作用,可能是消除肿瘤的关键。
近年来,从天然植物中寻找毒副作用小、安全有效的抗肿瘤药物成为研究的热点。金雀异黄素是来源于豆类植物与齿状植物的异黄酮类化合物,其结构与雌激素相似,并具有雌激素效能。流行病学调查发现与激素代谢密切相关的肿瘤如乳腺癌、前列腺癌等均和大豆制品的摄入呈负相关。大豆中许多成分(如大豆皂甙、植酸和蛋白酶抑制剂等)均有抗癌作用,其中所含的异黄酮特别是金雀异黄素可能是抗肿瘤作用的主要活性成分。金雀异黄素对人胃癌、食管癌、结直肠癌等细胞株均有不同程度的抑制作用。低浓度(<10μg/ml)表现为抑制细胞生长;高浓度(≥20μg/ml)除抑制肿瘤细胞生长外尚表现细胞毒作用,出现染色质浓缩,核碎裂和DNA断裂等细胞凋亡的结构变化。其抗肿瘤的作用机制主要包括:1、抑制酪氨酸蛋白激酶(PTK)的活性;2、增加抗氧化酶活性;3、雌激素及抗雌激素样作用;4、诱导细胞程序性死亡。5、抑制血管形成等。
以往的研究认为肝癌中有33%可查出雌激素受体,使用抗雌激素的三苯氧胺治疗肝癌已有报道。为探讨植物雌激素金雀异黄素临床应用于治疗肝癌的可行性,本研究运用MTT法、流式细胞仪检测、电镜观察、免疫组织化学染色、Western blot等实验手段和方法,针对金雀异黄素在抑制肝癌细胞增殖、促进凋亡、抗侵袭及其作用机制,进行了研究和探讨。
本实验发现金雀异黄素明显抑制人肝癌细胞SMMC-7721的生长,随药物浓度、作用时间延长,细胞增殖速度减慢,呈剂量-效应关系。当Gen浓度达到10.0μg /ml后,明显抑制了细胞的生长,10.0μg /ml 浓度时24h,48h的抑制率分别为58.8﹪,65.2﹪;Gen浓度为20.0μg /ml时, 24h细胞抑制率为78.8﹪,48h细胞抑制率达到了85.4﹪,与空白对照组有显著差异(P<0.01)。
流式细胞仪分析未用药组细胞G0/G1期所占比例最高,为60.56﹪,S期所占比例为30.17﹪,G2/M期细胞比例最少,仅为9.23﹪。随着Gen浓度从2.5μg /ml增加到20.0μg/ml的增加,作用24h后G2/M期细胞比例从9.23﹪增加到15.54﹪;作用48h后G2/M期细胞比例从8.56﹪增加到18.41﹪。结果显示随着Gen浓度的增加, G2/M期细胞增加,而S期细胞减少, G0/G1期比例也有减少趋势,二者比较有统计学意义(P<0.01)。本实验结果显示Gen通过影响细胞周期分布而影响细胞的代谢和功能,导致细胞重新分布,达到抑制细胞增殖的目的,这可能是其抗肿瘤机制的一方面。
透射电镜观察发现未用药组SMMC-7721细胞结构清楚,细胞膜表面有丰富的微绒毛。核膜完整,细胞核呈不规则形、圆形、椭圆形,核仁明显,核内染色质分布均匀。胞质内可见较多糖原颗粒,高尔基复合体、粗面内质网、线粒体等细胞器清楚。Gen浓度为5.0μg /ml作用细胞24h后,细胞核内染色质趋边凝集,或核内染色质成块状凝集。胞质内线粒体肿胀、空化,胞质轻度空化。Gen浓度为10.0μg /ml作用细胞24h后,细胞核呈固缩状改变,核膜不清晰,细胞核和细胞质呈空化状改变,线粒体空化,胞质基质空化。Gen浓度为20.0μg /ml作用细胞24h后,细胞核呈固缩状,核内异染色质趋边凝集,胞质电子密度加深,细胞质空化明显,并可见凋亡小体,呈典型的凋亡细胞的形态学改变,证实其抗癌的作用机制可能是诱导细胞凋亡。
免疫组织化学显示用不同浓度Gen处理的SMMC-7721细胞48小时,Caspases-3蛋白、Bax蛋白表达随着Gen浓度的增加,呈现上升趋势。10.0μg/ml和20.0ug/mlGen浓度组与对照组相比,Caspases-3蛋白、Bax蛋白阳性的细胞的阳性率差别有显著性(P<0.01)。P53和Bcl-2蛋白表达随着Gen浓度的增加,呈现下降趋势。10.0μg/ml和20.0μg /mlGen浓度组与对照组相比, P53、Bcl-2蛋白阳性的细胞的阳性率差别有显著性(P<0.01)。我们的研究结果显示金雀异黄素使突变型p53,bcl-2蛋白表达均下降,这说明金雀异黄素对二者表达均有抑制,考虑是其诱导凋亡的机制之一;Caspases-3,Bax蛋白表达增强,则可以促进肝癌细胞凋亡。但对哪一种蛋白作用更强,我们的试验并没有显著性差异。并且四种蛋白之间的相互作用在凋亡发生的机制中也是相当重要,而金雀异黄素对这四个蛋白的作用哪一个占主要地位,其作用的具体机制,还需进一步观察研究。
Western blot结果显示金雀异黄素可以调控人肝癌细胞MMP-2、MMP-9、TIMP-2、NF-κB表达,随着Gen浓度的增加,MMP-2、MMP-9、NF-κB表达下调,TIMP-2表达上调,证明金雀异黄素可能通过影响MMP-2、MMP-9、TIMP-2、NF-κB的表达,抑制人肝癌细胞的侵袭。
综上所述,金雀异黄素对人肝癌细胞SMCC-7721增殖有抑制作用并诱导凋亡,随浓度的增加及作用时间的延长抑制人肝癌细胞SMCC-7721增殖及诱导凋亡作用增强。影响细胞周期分布、诱导细胞凋亡可能是金雀异黄素抑制肝癌的主要机制之一。 体外实验表明,金雀异黄素可以调控人肝癌细胞MMP-2、MMP-9、TIMP-2、NF-κB表达,证明金雀异黄素在一定程度上可以抑制人肝癌细胞的侵袭。
金雀异黄素在肿瘤的生物治疗中具有广阔的临床应用前景,本实验为金雀异黄素在临床上的应用,提供了理论和实验依据。
关键词:金雀异黄素 肝癌



Hepatic cell carcinoma (HCC) is one of the frequent malignant tumors in China, with an annual mortality rate as high as 14.52 per ten thousand population. It is often accompanied with hepatic cirrhosis and propagates within liver at the early stage. Because of the lack of specific symptoms and signs, it is difficult to be detected early. Furthermore, its development is very rapid, so many patients miss the chance of surgery treatment. Ninety five percent patients are not sensitive to radiotherapy. Systemic chemotherapy has a poor curative effect, which is lower than fifteen percent. All these above-mentioned matters contribute to the rapid death of patients shortly after the onset of the disease. Therefore, it becomes an important research direction to search for drugs that can inhibit the growth of tumor cells and promote apoptosis but with low toxicity. Hino et al confirmed that apoptosis existed in all the development stages of hepatoma, and with the progression its growth rate is higher than apoptosis rate. Apoptosis is an active death procedure programmed by genes. Many works showed that apoptosis is closely associated with the pathogenesis and progression of hepatoma. For this reason, it appears to become the key point to find drugs that can induce apoptosis of neoplasm cells and have low toxicity to normal cells.
Recently, it has become a hot point to search for effective but low toxic drugs from natural plants. Genistein is a sort of osajin compound derived from bean and denticulatum plants, whose structure and potency are similar to that of estrogen. Epidemiological survey suggests that both breast cancer and prostatic carcinoma are negatively associated with the consumption of soybean products. Many components in soybean (for example soyasaponin, phytic acid and proteinase inhibitor) have anticancer effect. Isoflavone, especially genistein maybe are the main active component which has the anticancer effect. Genistein has depressant effects on cell lines of human gastric cancer, esophageal carcinoma and colorectal cancer in different degree. In low concentration(<10μg/ml), genistein shows inhibitory effect in eifferent degree against cancer; while in high concentration(>20μg/ml), it shows cytotoxic effect, resulting in apoptotic structural changes such as chromatin condensation, nuclear fragmentation and DNA interruption. The major anticancer mechanism of genistein lies in the following aspect: 1 inhibiting the activity of tyrosine protein kinase (PTK);2 increasing the activity of antioxidase;3 estrogen and antiestrogen-like effect; 4inducing programmed cell death; 5 inhibiting angiopoiesis.
In order to search for the possibility of applying genistein to the treatment of cancer, this study adopted the MTT reduction assay (MTT), flow cytometry, electron microscope, immunohistochemistry and Western blot to investigate the mechanism of genistein inhibiting hepatoma carcinoma cell proliferation, promoting apoptotic and blocking invasion.
This study discovered that genistein could notably inhibit the growth of hepatoma carcinoma cell SMMC-7721.With the increase of drug concentration and the duration of action time, the cell proliferation speed stepped down, showing dose-effect relationship. When the concentration reached 10.0μg /ml, cell growth was remarkably inhibited. The inhibition ratio was 58.8﹪ and 65.2﹪ respectively 24 hours and 48 hours after genistein adoption when the drug concentration was 10.0μg /ml. While when the drug concentration reached 20.0μg /ml, the inhibition ratio was 78.8﹪ and 85.4﹪ respectively 24 hours and 48 hours after genistein adoption, the difference was significant compared with that of normal control.
Flow cytometry analysis showed that the G0/G1 ratio was the highest in non-drug treatment group among all groups, reaching 60.56﹪;the ratio in S stage was 30.17﹪,the ration in G2/M stage was the lowest, only 9.23﹪.With the increment of genistein concentration from 2.5μg /ml to 20.0μg/ml, the ratio of G2/M raised from 9.23﹪ to 15.54﹪ after treatment of 24 hours, and raised from 8.56﹪ to 18.41﹪ after treatment of 48 hours. These results indicated that with the increment of genistein concentration, the cells in G2/M stage increased, cells in S stage decreased and the ratio of G0/G1 having the trend of decrement.
Transmission electron microscope analysis showed that SMMC-7721 cells in non-drug treatment group had clear cellular structure, having rich microvilli in cellular membrane and intact nuclear membrane. The cellular nucleus was irregular, round or ellipse, with clear nucleoli. Chromatin was well-distributed within the nucleus. Several glycogen granule could been seen in intracytoplasm . Cell organelles such as Golgi complex, rough endoplasmic reticulum and chondriosome were clear. After being treated with genistein 24 hours at the concentration of 5.0μg /ml, chromatin in cellular nucleus became conglomeration or lumping, with chondriosome swelling, cavitating and kytoplasm cavitating. After being treated with genistein at the concentration of 10.0μg /ml 24 hours, cellular nucleus became condensed, nuclear membrane unclear, cytoblast and cytoplasm cavitating, ground cytoplasm cavitating. Twenty four hours after being treated with genistein at the concentration of 20.0μg /ml, cellular nucleus became condensed, heterochromatin agglutinating, kytoplasm electron density deepening, cytoplasm obviously cavitating, apoptotic body appearing, showing typical apoptosis changes in morphology, indicating that the anticancer mechanism of genistein involving the induction of cell apoptosis.
Immunohistochemistry results showed that SMMC-7721 cell after being treated 48 hours with different concentration of genistein, the expression of Caspases-3 and Bax protein increased with the increment of genistein concentration.The positive rate of Caspases-3 and Bax protein in cells of 10.0μg/ml and 20.0ug/ml genistein concentration group was remarkably different from that in normal control group. The expression of p53 and Bcl-2 protein expression had the tendency to drop with the increment of genistein concentration. Compared with control group, positive rates of p53 and Bcl-2 protein expression in cells of 10.0μg/ml and 20.0μg /ml genistein concentration groups were significantly different from that in control group.
Results of Western blot showed that genistein could regulate the expression of MMP-2,MMP-9,TIMP-2 and NF-κB in human hepatoma carcinoma cells. With the increment of genistein concentration, the expression of MMP-2,MMP-9 and NF-κB up-regulated and TIMP-2 down-regulated, showing that genistein probably inhibited the invision of human hepatoma carcinoma cells by means of influencing the expression of MMP-2,MMP-9,TIMP-2 and NF-κB.
Genistein has a promising applying prospect in the field of biotherapy of tumors.The research provides a theoretical and experimental foundation for the clinically applying genistein against tumors.
Key word: Genistein hepatoma