The 5th Department of General Surgery, Lanzhou University Second Hospital, Lanzhou, Gansu 730030, China.
Department of Pharmacy, Lanzhou University Second Hospital, Lanzhou, Gansu 730030, China.
Chin Med J (Engl). 2019 May 5;132(9):1071-1078. doi: 10.1097/CM9.0000000000000199.
Colorectal cancer is the third most common cancer worldwide and still lack of effective therapy so far. Petasin, a natural product found in plants of the genus Petasites, has been reported to possess anticancer activity. The present study aimed to investigate the anticolon cancer activity of petasin both in vitro and in vivo. The molecular mechanism of petasin was also further explored.
Caco-2, LoVo, SW-620, and HT-29 cell lines were used to detect the inhibitory effect of petasin on colon cancer proliferation. Cell viability was determined using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay. Cell apoptosis was analyzed by flow cytometry. Hoechst 33258 staining was used to visualize morphological changes. Cell migration was assessed using a wound-healing migration assay, and cell invasion was investigated using Transwell chambers. Western blotting assays were employed to evaluate the expression levels of proteins in the protein kinase B/mammalian target of rapamycin (Akt/mTOR) signaling pathway. Finally, in vivo activity of petasin was evaluated using the SW-620 subcutaneous tumor model established in Balb/c nude mice. Twelve rats were randomly divided into control group and 10 mg/kg petasin group. The tumor volume was calculated every 7 days for 28 days. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay was performed to assess the apoptotic effect of petasin. Differences between two groups were assessed by analysis of independent-sample t tests.
Petasin significantly inhibited the proliferation of human colon carcinoma cell lines, induced apoptosis, and suppressed migration and invasion in SW-620 cells. Western blotting results showed that petasin decreased the phosphorylation of Akt (1.01 ± 0.16 vs. 0.74 ± 0.06, P = 0.042), mTOR (0.71 ± 0.12 vs. 0.32 ± 0.11, P = 0.013), and P70S6K (1.23 ± 0.21 vs. 0.85 ± 0.14, P = 0.008), elevated the expression of caspase-3 (0.41 ± 0.09 vs. 0.74 ± 0.12, P = 0.018) and caspase-9 (1.10 ± 0.27 vs. 1.98 ± 0.22, P = 0.009), decreased the Bcl-2 protein (2.75 ± 0.47 vs. 1.51 ± 0.36, P = 0.008), downregulated the expression of matrix metalloproteinase (MMP)-3 (1.51 ± 0.31 vs. 0.82 ± 0.11, P = 0.021) and MMP-9 (1.56 ± 0.32 vs. 0.94 ± 0.15, P = 0.039) in SW-620 cell. In vivo, 10 mg/kg petasin inhibited tumor growth in Balb/c nude mice (924.18 ± 101.23 vs. 577.67 ± 75.12 mm at day 28, P = 0.001) and induced apoptosis (3.6 ± 0.7% vs. 36.0 ± 4.9%, P = 0.001) in tumor tissues.
Petasin inhibits the proliferation of colon cancer SW-620 cells via inactivating the Akt/mTOR pathway. Our findings suggest petasin as a potential candidate for colon cancer therapy.
结直肠癌是全球第三大常见癌症，迄今为止仍然缺乏有效的治疗方法。芹菜素是一种存在于款冬属植物中的天然产物，已被报道具有抗癌活性。本研究旨在探讨芹菜素在体外和体内对结肠癌的抗癌活性。此外，还进一步探讨了芹菜素的分子机制。
使用 Caco-2、LoVo、SW-620 和 HT-29 细胞系检测芹菜素对结肠癌增殖的抑制作用。通过 MTT（3-[4,5-二甲基噻唑-2-基]-2,5-二苯基四唑溴盐）测定法测定细胞活力。通过流式细胞术分析细胞凋亡。使用 Hoechst 33258 染色观察形态学变化。通过划痕愈合迁移实验评估细胞迁移，通过 Transwell 室评估细胞侵袭。采用 Western blot 法检测蛋白激酶 B/哺乳动物雷帕霉素靶蛋白（Akt/mTOR）信号通路中蛋白的表达水平。最后，在 Balb/c 裸鼠建立的 SW-620 皮下肿瘤模型中评估芹菜素的体内活性。将 12 只大鼠随机分为对照组和 10mg/kg 芹菜素组。在 28 天内每 7 天计算一次肿瘤体积。使用末端脱氧核苷酸转移酶介导的 dUTP 缺口末端标记（TUNEL）实验评估芹菜素的凋亡作用。通过分析独立样本 t 检验评估两组间的差异。
芹菜素显著抑制人结肠癌细胞系的增殖，诱导细胞凋亡，并抑制 SW-620 细胞的迁移和侵袭。Western blot 结果显示，芹菜素降低了 Akt 的磷酸化（1.01±0.16 对 0.74±0.06，P=0.042）、mTOR（0.71±0.12 对 0.32±0.11，P=0.013）和 P70S6K（1.23±0.21 对 0.85±0.14，P=0.008），上调了 caspase-3（0.41±0.09 对 0.74±0.12，P=0.018）和 caspase-9（1.10±0.27 对 1.98±0.22，P=0.009）的表达，降低了 Bcl-2 蛋白（2.75±0.47 对 1.51±0.36，P=0.008），下调了基质金属蛋白酶（MMP）-3（1.51±0.31 对 0.82±0.11，P=0.021）和 MMP-9（1.56±0.32 对 0.94±0.15，P=0.039）的表达。在体内，10mg/kg 芹菜素抑制 Balb/c 裸鼠的肿瘤生长（第 28 天的 924.18±101.23 对 577.67±75.12mm，P=0.001）并诱导肿瘤组织中的细胞凋亡（3.6±0.7%对 36.0±4.9%，P=0.001）。
芹菜素通过抑制 Akt/mTOR 通路抑制结肠癌细胞 SW-620 的增殖。我们的研究结果表明芹菜素可能是治疗结肠癌的潜在候选药物。
