Castro-Carvalho Bruno, Ramos Alice A, Prata-Sena Maria, Malhão Fernanda, Moreira Márcia, Gargiulo Daniela, Dethoup Tida, Buttachon Suradet, Kijjoa Anake, Rocha Eduardo
Interdisciplinary Center for Marine and Environmental Research, University of Porto, 4450-208 Matosinhos, Portugal.
Department of Microscopy, Institute of Biomedical Sciences Abel Salazar, University of Porto, 4050-313 Porto, Portugal.
Pharmacognosy Res. 2017 Dec;9(Suppl 1):S92-S98. doi: 10.4103/pr.pr_57_17.
Drug resistance is a major concern in the current chemotherapeutic approaches and the combination with natural compounds may enhance the cytotoxic effects of the anticancer drugs. Therefore, this study evaluated the cytotoxicity of crude ethyl extracts of six marine-derived fungi - KUFC 9213 (E1), KUFC 7896 (E2), KUFC 6344 (E3), KUFA 0013 (E4), KUFC 7894 (E5), and KUFC 0021 (E6) - when combined with doxorubicin (Dox), in seven human cancer cell lines.
The antiproliferative activity was primarily assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay.
Two extracts, E1 and E2, demonstrated a significant enhancement of Dox's cytotoxicity in nonsmall cell lung cancer A549 cells. Accumulation of Dox in the nuclei increased when A549 cells were treated in combination with extracts E1 and E2, with induction of cell death observed by the nuclear condensation assay. The combination of E2 with Dox increased the DNA damage as detected by the comet assay. Ultrastructural observations by transmission electron microscopy suggest an autophagic cell death due to an increase of autophagic vesicles, namely with the combination of Dox with E1 and E2.
These findings led to the conclusion that the fungal extracts E1 and E2 potentiate the anticancer action of Dox, through nuclear accumulation of Dox with induction of cell death mainly by cytotoxic autophagy.
Fungal extracts increase the cytotoxic activity of doxorubicin (Dox) in lung cancer cellsNuclear accumulation of Dox, DNA damage, and cell death as a mechanism of actionFungal extracts may potentiate the anticancer activity of conventional drugs. A375: Human malignant melanoma cell line, A549: Human non small lung cancer cell line, DAPI: 4,6-Diamidino-2-phenylindole, DMEM: Dulbecco's Modified Eagle Medium, DMSO: Dimethylsulfoxide, Dox: Doxorubicin, DSBs: DNA double-strand breaks, E1: KUFC 9213, E2: KUFC 7896, E3: KUFC 6344, E4: KUFA 0013, E5: KUFC 7894, E6: KUFC 0021, FBS: Fetal bovine serum, HCT116: Human colorectal carcinoma cell line, HEPES: (N-[2-hydroxyethyl] piperazine-N'- [2-ethane-sulfonic acid]), HepG2: Human hepatocellular carcinoma cell line, HT29: Human Caucasian colon adenocarcinoma Grade II cell line, IC: Concentration of the extract or Dox that inhibits cell viability by 50%, LRP: Lung resistance-related protein, MCF7: Human breast adenocarcinoma cell line, MEM: Minimum Essential Medium Eagle, MRPs: Multidrug resistance-associated proteins, MTT: 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, PBS: Phosphate-buffered saline, NSCLC: Nonsmall cell lung cancer, P-gp: P-glycoprotein, ROS: Reactive oxygen species, RPMI: Roswell Park Memorial Institute Medium, TEM: Transmission electron microscopy, U251: Human glioblastoma astrocytoma cell line.
耐药性是当前化疗方法中的一个主要问题，与天然化合物联合使用可能会增强抗癌药物的细胞毒性作用。因此，本研究评估了六种海洋来源真菌的粗提物——KUFC 9213（E1）、KUFC 7896（E2）、KUFC 6344（E3）、KUFA 0013（E4）、KUFC 7894（E5）和KUFC 0021（E6）——与阿霉素（Dox）联合使用时对七种人类癌细胞系的细胞毒性。
主要通过3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐法评估抗增殖活性。
提取物E1和E2在非小细胞肺癌A549细胞中显著增强了阿霉素的细胞毒性。当A549细胞与提取物E1和E2联合处理时，细胞核中阿霉素的积累增加，通过核凝聚试验观察到细胞死亡诱导。彗星试验检测显示，E2与阿霉素联合增加了DNA损伤。透射电子显微镜的超微结构观察表明，由于自噬小泡增加，即阿霉素与E1和E2联合时，导致自噬性细胞死亡。
这些发现得出结论，真菌提取物E1和E2通过阿霉素的核积累并主要通过细胞毒性自噬诱导细胞死亡来增强阿霉素的抗癌作用。
真菌提取物增加阿霉素（Dox）在肺癌细胞中的细胞毒性活性
阿霉素的核积累、DNA损伤和细胞死亡作为作用机制
真菌提取物可能增强传统药物的抗癌活性。A375：人恶性黑色素瘤细胞系，A549：人非小细胞肺癌细胞系，DAPI：4,6-二脒基-2-苯基吲哚，DMEM：杜氏改良伊格尔培养基，DMSO：二甲基亚砜，Dox：阿霉素，DSBs：DNA双链断裂，E1：KUFC 9213，E2：KUFC 7896，E3：KUFC 6344，E4：KUFA 0013，E5：KUFC 7894，E6：KUFC 0021，FBS：胎牛血清，HCT116：人结肠癌细胞系，HEPES：（N-[2-羟乙基]哌嗪-N'-[2-乙烷磺酸]），HepG2：人肝癌细胞系，HT29：人高加索结肠腺癌II级细胞系，IC：抑制细胞活力50%的提取物或阿霉素浓度，LRP：肺耐药相关蛋白，MCF7：人乳腺腺癌细胞系，MEM：伊格尔最低必需培养基，MRPs：多药耐药相关蛋白，MTT：3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐，PBS：磷酸盐缓冲盐水，NSCLC：非小细胞肺癌，P-gp：P-糖蛋白，ROS：活性氧，RPMI：罗斯威尔帕克纪念研究所培养基，TEM：透射电子显微镜，U251：人胶质母细胞瘤星形细胞瘤细胞系。
