Anticancer Activity of Modified Tongyou Decoction on Eca109 Esophageal Cancer Cell Invasion and Metastasis through Regulation of the Epithelial-Mesenchymal Transition Mediated by the HIF-1 α-Snail Axis

Abstract

Background: To explore the activity of Modified Tongyou Decoction (MTD) against Eca109 esophageal cancer (EC) cell invasion and metastasis and to ascertain the mechanism of its anticancer activity during the epithelial-mesenchymal transition (EMT) as mediated by the HIF-1α-Snail axis.

Methods: Herbal compounds were prepared by ethanol extraction, and 6 herbs composing into MTD were dipped in water-free ethanol and filtered. The filtrate was collected and centrifuged. The remains were concentrated into a paste which was adjusted to 5000mg/mL concentration with DMSO. PBS was used to dilute the herbal solution to the half maximal inhibitory concentration. A hypoxic microenvironment was induced with CoCl₂ in RPMI 1640 medium, in which Eca109 cells were cultured. The cytotoxicity of MTD was determined with CCK-8 assay. The activity of MTD against cell invasion and metastasis was explored with scratch assay and transwell assay. Western blot analysis was conducted to analyze the anticancer effects of MTD on the expression of HIF-1α-Snail axis- and EMT-related proteins. Quantitative RT-PCR was used to assess the mRNA expression of Snail. Immunofluorescence labeling was performed to examine how MTD affected the coexpression of Snail and HIF-1α.

Results: The fifty percent inhibitory dose of MTD was 1410 μg/mL in the normoxic environment and 1823 μg/mL in the hypoxic environment based on the CCK-8 assay. The scratch assay showed that MTD significantly inhibited cell migration in both the normoxic and hypoxic microenvironments compared with the control groups (P < 0.05). The transwell assay showed that MTD significantly inhibited cell invasion in both the normoxic and hypoxic environments compared with the control groups (P < 0.05). Western blot showed that MTD significantly inhibited the expression of the HIF-1α, Snail, Vimentin, MMP-2, MMP-9, and VE-cadherin proteins and significantly induced the expression of E-cadherin in both the normoxic and hypoxic microenvironments compared with the control groups (P < 0.05). qRT-PCR indicated that MTD significantly inhibited Snail mRNA expression compared with that in the control groups (P < 0.05). Immunofluorescence assay showed that MTD significantly inhibited the coexpression of HIF-1α and Snail in both the normoxic and hypoxic microenvironments compared with the control groups (P < 0.05).

Conclusion: MTD downregulated HIF-1α-Snail axis- and EMT-related proteins to inhibit EC cell invasion and metastasis in both the normoxic and hypoxic environments.

Figure 1

MTD inhibited Eca109 cells proliferation in a direct dose-effect manner in both normoxia and hypoxia. Notes: Relation between MTD concentration and cell inhibition rate showed the same tendency in both normoxia and hypoxia, fitted curve taking on cubic manner. (a) Dose-effect manner in normoxia: inhibition rate reached peak at 2560 μg/mL, equal to the rate at 5120 μg/mL, fitted formula, Y(%) = 9.738836 + 0.064047x + (−2 × 10⁻⁵)x² + (1.93 × 10⁻⁹)x³. (b) Dose-effect manner in hypoxia, inhibition rate reached peak at 5120 μg/mL, fitted formula, Y(%) = 9.255399 + 0.061135x + (−2.2 × 10⁻⁵)x² + (2.48 × 10⁻⁹)x³.

Figure 2

MTD inhibited Eca109 cell migration in both normoxia and hypoxia.

Figure 3

MTD inhibited Eca109 cell invasion in both normoxia and hypoxia. Note: (a) in Group OC, transwell cells were in vigorous proliferation; in Group OM, cells were infertile, showing light color; in Group HC, cells were in mass and proliferating exuberant; in Group HM, cells were inhibited greatly and mass disappeared. (b) In Group OM, cell number reduced remarkably, showing statistical difference from that in Group OC, ^∗∗P < 0.05. What is noteworthy, in Group HC, cell number increased greatly, showing statistical difference from that in Group OC, ^∗∗P < 0.05. In Group HM, cell number reduced sharply, showing statistical difference from that in Group HC, ^##P < 0.05. Homogeneity of variance by one-way ANOVA; LSD test applied to compare statistical difference.

Figure 4

Protein expression of the HIF-1α-Snail axis and EMT-related proteins. Note: A, Group OC; B, Group OM; C, Group HC; D, Group HM. Compared with Group OC, ^∗∗P < 0.05; compared with Group HC, ^##P < 0.05.

Figure 5

MTD inhibited Snail mRNA expression in both normoxia and hypoxia. Note: (a) amplification curve, as the target gene, Snail was measured with GAPDH. Value of Ct was in the domain of 15-30, and ▵Rn was in the domain of 0.5–1.5. (b) In Group OM, mRNA expression of Snail was inhibited remarkably, showing statistical difference from that in Group OC, ^∗∗P < 0.05. What is noteworthy, in Group HC, Snail mRNA overexpressed greatly, showing statistical difference from that in Group OC, ^∗∗P < 0.05. In Group HM, the mRNA expression was inhibited significantly, showing statistical difference from that in Group HC, ^##P < 0.05. Homogeneity of variance by one-way ANOVA; LSD test applied to compare statistical difference.

Figure 6

MTD inhibited Snail and HIF-1α fluorescent signals in both normoxia and hypoxia (200x). Note: blue fluorescence was DAPI, the green labeled Snail and HIF-1α, respectively. “Merge” was the combination of blue and green signals.

Figure 7

Relative OD values of HIF-1α and Snail fluorescent signals inhibited by MTD in both normoxia and hypoxia Note: in Group OM, HIF-1α and Snail fluorescent signals weakened remarkably, showing statistical difference from those in Group OC, ^∗∗P < 0.05. What is noteworthy, in Group HC, both signals strengthened greatly, showing statistical difference from those in Group OC, ^∗∗P < 0.05. In Group HM, both signals weakened significantly, showing statistical difference from those in Group HC, ^##P < 0.05. Homogeneity of variance by one-way ANOVA; LSD test applied to compare statistical difference