DMH1 effectively inhibited CT-26 cell migration under hypoxia conditions
The in vitro experiments revealed that DMH1 stimulated CT-26 cell apoptosis at a concentration of more than 5mM after 24 h. However, it had no effect on the apoptosis of CT-26 cells at a concentration of less than 1mM (Fig. 1a) with or without serum. Hence, 1mM DMH1 was used to examine whether DMH1 inhibited the migration of CT-26 cells.
The results indicated that the number of CT-26 cells that migrated through the chamber membrane markedly reduced after treatment with DMH1 under hypoxic conditions (P < 0.01, Fig. 1b). But DMH1 had no effect on the migration of CT-26 cells under normxia (Fig. 1b).
DMH1 effectively inhibited CT-26 cell migration via HIF-1α
Previous studies demonstrated that DMH1 suppressed migration [7]. However, the underlying mechanism was still unknown.
HIF-1α regulates the expression of several genes involved in cancer cells migration under hypoxia conditions [3]. In our studies, we found that treatment with DMH1 significantly downregulate the high level of HIF-1α under hypoxic conditions (P < 0.01, Fig. 2a). And hence, we wondered that DMH1 suppress migration of CT-26 through HIF-1α mediate signaling pathway.
Incubating CT-26 cells with CoCl2 (the specific inducer of HIF-1α, 150μM) significantly increased the protein levels of HIF-1α (Fig. 2b), but not those of HIF-1β (data not shown). The transwell assay results showed that treatment with CoCl2 stimulated CT-26 cell migration. However, co-application of CoCl2 and DMH1 inhibited the CoCl2-induced migration of CT-26 cells (P < 0.01, Fig. 2c-d). Next, the HIF-1α siRNA (siHIF-1α) was used to test the hypothesis. The protein level of HIF-1α decreased in CT-26 cells after the transfection of HIF-1α siRNA, but not that of HIF-1β siRNA (data not shown), compared with that in the non-transfection group (P <0 .01, Fig. 3a). These data indicated that the experimental conditions were reliable.
Next, the Transwell assay was used to clarify the effects of DMH1 incubation on HIF-1α-induced cell migration. The results revealed that CoCl2 stimulated the migration and DMH1 inhibited the migration of CT-26 cells in the negative control (NC) group. However, CoCl2 did not stimulate the migration of CT-26 cells after transfection of HIF-1α siRNA. Also, siRNA-mediated HIF-1α knockdown almost completely abrogated the inhibition of migration induced by incubation with DMH1 (P < 0.01, Fig. 3b). These results accorded with the results obtained after culturing CT-26 cells transfected with HIF-1α siRNA under hypoxia condition (P < 0.01, Fig. 3c). And hence, those results indicated that DMH1 inhibit the migration of CT-26 cells via HIF-1α.
DMH1 suppressed lactate-mediated CT-26 cell migration
Recently, some studies confirmed that the accumulation of lactate reduced the pH in the tumor microenvironment, which induced cancer cell migration [9]. Hence, it was hypothesized that increased lactate levels in culture media, which reduced the extracellular pH (pHe), might stimulate the migration of cancer cells.
In our studies, we found that the CT-26 cells cultured at pH 6.8 migrated much more compared with cells grown at pH 7.4 (tested at pH 6.8). Accordingly, CT-26 cells grown at pH 7.4 migrated less compared with those cultured at pH 6.8 (tested at pH 7.4). These findings supported that low pH (acidic environment) stimulated the migration of cancer cells (Fig. 4a).
Furthermore, it was found that exposure to 20mM lactate for 24 h was not cytotoxic, irrespective of the presence or absence of serum. Hence, 20mM lactate was chosen for the subsequent experiments (Fig. 4b).
After incubation with lactate for 24 h, it was found that the pH in the upper compartment was lower than that in the lower compartment when lactate was added to the upper compartment. Also, the pH in the lower compartment was lower than that in the upper compartment when lactate was added to the lower compartment. These results confirmed that lactate could not enter the membrane from upper to lower (or lower to upper) compartments (Fig. 4c). The Transwell assay showed that 20mM lactate delivered into the upper compartment significantly stimulated CT-26 cell migration, but lactate did not act as a chemoattractant (Fig. 4c) [10].
The observed migratory changes could be exclusively not due to an unspecific cellular response. Hence, the migratory effects were tested by adding D-lactate, NaCl, or pyruvate. Initially, adding D-lactate (20mM) did not affect CT-26 cell migration. It was verified that the changes in cell migration could not be attributed to the changes in osmolarity after adding sodium chloride (NaCl, 20mM), similar to D-lactate. Pyruvate (20mM) also did not promote CT-26 cell migration (Fig. 4d). Therefore, the results confirmed that L-lactate promoted CT-26 cell migration.
DMH1 inhibited CT-26 cell migration through HIF-1α/MCT-4 signaling pathway
Our previous studies indicated that 1) low extracellular pH stimulate the migration of CT-26 cells which induce by lactate; 2) DMH1 inhibit the migration of CT-26 cells via HIF-1α. But, whether DMH1 inhibited the migration of CT-26 cells via HIF-1α induce the excretion of lactate was still unknown.
In our experiments, the lactate concentration in the culture media was upregulated over time (P < 0.01; Fig. 5a). However, the lactate level was downregulated in the culture media with CT-26 cells after treatment with DMH1 under hypoxic conditions and treatment with CoCl2. The same results were also obtained after transfection of HIF-1α siRNA (P < 0.01, Fig. 5a).
Our study indicated that DMH1 inhibited lactate excretion in CT-26 cells under hypoxic conditions. However, whether DMH1 affected lactate-induced extracellular pH alteration (pHe) remained unknown. The CT-26 cells underwent extracellular acidification within 24 h after hypoxia exposure and treatment with CoCl2 (P < 0.01, Fig. 5b). However, treatment with DMH1 significantly reversed the low extracellular pH which induce by hypoxia and CoCl2. The same results were also obtained after transfection of HIF-1α siRNA.
Studies indicated that MCT-4 regulated the excretion of lactate in the tumor microenvironment, which always accompanied extracellular acidification [8]. And the Long-term exposure of tumor cells under extracellular acidification leads to migration [16]. Under normxia, DMH1 had no effect on the expression of MCT-4(Fig 5c). However, the protein levels of MCT-4 was upregulated under hypoxia and treatment with CoCl2, and DMH1 downregulated the high protein level of MCT-4 under hypoxia and CoCl2 (P < 0.01, Fig. 5d-e). Surely, the transfection of HIF-1α siRNA suppressed the high level of MCT-4 under hypoxia (P < 0.01, Fig. 5d-e). The same results were also obtained after transfection of HIF-1α siRNA.
Studies confirmed that MCT-4 is one of the downregulators of HIF-1α [8]. And our results also indicated that DMH1 inhibited the extracellular acidification through HIF-1α/MCT-4 induced excretion of lactate in CT-26 cells. But, whether DMH1 inhibited the migration of CT-26 cells via MCT-4 induce the excretion of lactate was still unknown. With RNAi assay, we found MCT-4 levels decreased in cultured CT-26 cell transfected with MCT-4 siRNA compared with that in the non-transfection group (P < 0.01, Fig. 6a).
In our studies, we also observed that CT-26 cells underwent extracellular acidification within 24 h after hypoxia exposure and treatment with CoCl2 (P < 0.01, Fig. 6b-c). Transfection of MCT-4 siRNA reversed the low extracellular pH which induce by hypoxia and CoCl2..
The transwell assay indicated that the transfection of MCT-4 siRNA inhibited CT-26 cell migration after treatment with CoCl2 (P < 0.01, Fig. 7a) . The same results were also observed under hypoxic conditions (P <0.01, Fig. 7b).