4-[6-(4-Isopropoxyphenyl)Pyrazolo[1,5-a]Pyrimidin-3-yl]Quinoline) Suppresses Metastasis Through HIF-1α/MCT-4-Mediated Lactate Excretion in Colorectal Cancer Cells

DMH1 (D4-[6-(4-isopropoxyphenyl)pyrazolo[1,5-a]pyrimidin-3-yl]quinoline) is a bone morphogenetic protein antagonist. However, the inuence of DMH1 on colorectal cancer metastasis remains unknown. Hence, This study aimed to investigate the inhibitory effect of DMH1 on the migration of cancer cells. In vitro incubation with DMH1(1mM) inhibited the high protein level of HIF-1α in CT-26 cells. Transwell assay conrmed that DMH1 suppress CT-26 cells migration via the HIF-1α under hypoxia. The accumulation of lactate in culture medium down-regulated the extracellular pH, then stimulated the migration of CT-26 cells. DMH1 suppressed the migration of CT-26 cells through HIF-1α/MCT-4 signaling pathway mediate the excreation of lactate. The data demonstrated that DMH1 downregulated the high intracellular pH levels via the HIF-1α/MCT-4 signaling pathway to mediate lactate excretion. This study indicated that DMH1 might serve as a potential drug for treating tumor migration by minimizing the intracellular pH via the HIF-1α/MCT-4 signaling pathway.


Introduction
Cancer metastasis is the primary cause of high mortality in patients with colorectal cancer [1]. It is considered one of the most important factors for patient survival [2].
Hypoxia is a common characteristic of tumors. The hypoxic response is primarily attributed to the hypoxia-inducible factor (HIF). HIF is composed of α-and β-subunits and plays a central role in adapting and responding to low oxygen levels. Studies have indicated that HIF-1α regulates the expression of several genes involved in cell metastasis under hypoxia conditions [3].
This study examined the effects of DMH1 on the expression of HIF-1α in CT-26 cells in vivo and in vitro, the effects of DMH1 on CT-26 cell migration, and the underlying mechanism. The ndings indicated that DMH1 inhibited cancer metastasis, thus providing preclinical evidence for using DMH1 to impede colon cancer cell migration.

Materials And Methods
Cell culture and cell transfection Page 3/17 CT-26 cells were maintained in RPMI-1640 medium (HyClone, UT, USA) supplemented with 10% fetal bovine serum (Gibco, CA,USA), 100 U/mL penicillin, and 100 mg/mL streptomycin (Gibco, CA, USA). All cell lines were cultured at 37°C in 5% CO 2 environment. The hypoxic condition was achieved by placing cancer cells in a sealed hypoxia chamber equilibrated with a triple gas mixture of 1% O 2 , 5% CO 2 and 94% N 2 .
Cell viability assay CT-26 cells were seeded into 96-well plates (5×10 5 cells/well) and incubated at 37°C for 24 h. On reaching 90% con uence, the cells were treated with different concentrations of DMH1 for 24 h. 20 μL of MTT (pH 4.7) was added to each well, and then the cells were incubated for another 4 h. 100 μL of 10% SDS 0.01 M HCl was added, and the cells were incubated at 37°C overnight to dissolve the formazan crystals.
Absorbance was measured at 570 nm.

Western blot
Brie y, the samples were subjected to electrophoresed in 10% SDS-PAGE and separated proteins were transfered to nitrocellulose membrane. The membrane were blocked in 5% non-fat milk overnight at 4℃.

Transwell assay
Cell migration assays were performed using 8.0 µm pore size coated with 0.5% gelatin (Costar). The lower chamber contained 15% serum as a chemoattractant. Cancer cells were prepared in serum-free medium, and 5×10 4 cells were added to the upper chamber in migration buffer (M199 containing 0.1% BSA). After 4 h of incubation at 37°C, cells were removed from the upper surface of the membranes with a cotton swab, and cells that migrated to the lower surface were xed with 4% paraformaldehyde for 30 mins and then stained with 0.1% crystal violet for 10 mins. Migrated cells were counted under a microscope.

Lactate concentration
The concentration of lactate in culture media were detected by using assay kits (Cat#A019-2-1, Nanjing Jiancheng Bioengineering Institute, China) according to the manufacturer's instructions.

Statistics
All data were expressed as mean ± standard error of the mean. Data were assessed with SPSS 15.0 evaluation version (SPSS, Chicago, IL, USA) statistical programme with one-way analysis of variance. P < 0.01 was considered indicate a significant.

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 signi cantly 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 CoCl 2 (the speci c inducer of HIF-1α, 150μM) signi cantly 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 CoCl 2 stimulated CT-26 cell migration. However, co-application of CoCl 2 and DMH1 inhibited the CoCl 2 -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 CoCl 2 stimulated the migration and DMH1 inhibited the migration of CT-26 cells in the negative control (NC) group. However, CoCl 2 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 con rmed 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 ndings 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 con rmed 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 signi cantly 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 unspeci c 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 veri ed 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 con rmed 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 CoCl 2 . 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 acidi cation within 24 h after hypoxia exposure and treatment with CoCl 2 (P < 0.01, Fig. 5b). However, treatment with DMH1 signi cantly reversed the low extracellular pH which induce by hypoxia and CoCl 2 . The same results were also obtained after transfection of HIF-1α siRNA. In our studies, we also observed that CT-26 cells underwent extracellular acidi cation within 24 h after hypoxia exposure and treatment with CoCl 2 (P < 0.01, Fig. 6b-c). Transfection of MCT-4 siRNA reversed the low extracellular pH which induce by hypoxia and CoCl 2 .. The transwell assay indicated that the transfection of MCT-4 siRNA inhibited CT-26 cell migration after treatment with CoCl 2 (P < 0.01, Fig. 7a) . The same results were also observed under hypoxic conditions (P <0.01, Fig. 7b).

Discussion
Migration is one of the most important aspects of the malignant cancer phenotype. Hence, clarifying the mechanisms of migration in cancer cells is important for developing new therapeutic strategies for cancer. The present study demonstrated the following: (1) DMH1 inhibited CT-26 cell migration via the HIF-1α/MCT-4 signaling pathway. (2) DMH1 reversed the extracellular acidi cation under hypoxic conditions, which was always associated with cancer migration.
Cancer cells deploy several strategies for migration and metastasis, which suggests that cancer cells need a special microenvironment for metastasis [11]. Protein analyses in animal models suggested that tumors expressed high levels of HIF-1α, indicating that they are in a sustained hypoxic environment. The results indicated that hypoxia was an important stimulant to initiate migration and metastasis [3]. Also, DMH1 inhibited migration in CT-26 cells after incubation with CoCl 2 and under hypoxic conditions in vitro.
These results indicated that DMH1 suppressed tumor migration via HIF-1α expression. It was found that DMH1 suppressed the high protein level of HIF-1α induced by CoCl 2 . DMH1 was found to regulate the expression of HIF-1α. However, the exact mechanism is still unknown. Some reports provided evidence that the negative feedback existed between PHD-2 and HIF-1α in cancer cell lines, indicating that PHD-2 might be one of the upstream regulators of HIF-1α [12]. However, several pathways have also been shown to control HIF-1α stability and transcriptional activity via post-translational modi cations under hypoxic conditions [13]. Therefore, further studies into the molecular mechanisms associated with the effects of DMH1 on the migration of cancer cells should explore its effect on the upstream regulation.
The present study also indicated that DMH1 did not induce apoptosis at lower doses but inhibited the migration of cancer cells at low doses in vitro. Hence, DMH1 was not a cytotoxic agent and might suppress the migration of cancer cells at lower doses. Also, it was speculated that DMH1 might serve as a potential anti-cancer agent. Some reports showed that treatment with DMH1 dramatically reduced lung tumor growth in subcutaneous xenograft mouse models [14]. They demonstrated that DMH1 increased the e cacy of anti-tumor drugs via blocking BMP signal [6]. However, studies indicated that the effects of DMH1 in attenuating tumor growth and migration might be mediated by multiple mechanisms. Hence, the possibility that DMH1 inhibited the migration of cancer cells through BMPs could not be excluded. Future studies should focus on whether DMH1 treatment may similarly disrupt the microenvironment required for tumor growth and migration [15].
Studies indicated that the excretion of lactate in the MCT-4-induced cancer microenvironment was always accompanied by extracellular acidi cation [16], and the long-term exposure of tumor cells under extracellular acidi cation led to migration [17]. Hence, it was proposed that the high lactate levels in tumors re ected the extent of the tumor cell adaptation to migration. The results indicated that the lactate concentration in cancer cell microenvironment increased, which was associated with a lower pHe after culture for 24 h.
The results indicated that the high level of lactate may induce the migration of CT-26 cells. However, lactate did not function as a chemoattractant in the studies. Many factors might affect the migration of cancer cells. Studies only showed that L-lactate, but not D-lactate, induced the migration of cancer cells osmotic effects, or metabolic activation [10]. Studies demonstrated the enhancement of the metastatic potential of tumors generally exhibiting pHe values of 6.7-6.8 [18]. Hence, it was speculated that the alteration of pHe and pHi might be one of the potential mechanisms that induced the migration of cancer cells [20]. Studies con rmed that tumor cells were highly e cient at maintaining plasmalemmal pH gradients, in which the extracellular pH (pHe) might be signi cantly lower than the pHi [19]. DMH1 reversed the extracellular acidi cation. The results indicated that the extracellular environment was more acidic than the intracellular environment (data not shown), leading to a pH gradient across the cell membrane. The results accorded with the previous nding that pH regulation was crucial in in uencing cancer cell metastasis [20]. The pHe/pHi gradient was downregulated (intracellular acidi cation occurred to lower the pH), which was dangerous to cancer development [21]. The pH alterations stimulated the migration of CT-26 cells; however, con icting results were reported in vitro [22]. Some studies found that the low pHe could stimulate the migration of cells [23]. Hence, the question arose whether the low pHeinduced modi cations of the microenvironment regulated by signal transduction might also affect the actin polymerization. Therefore, further studies should be conducted to detect (1) whether DMH1 regulates the pH of the tumor microenvironment and (2) whether DMH1 regulates the alteration of pH in the tumor microenvironment.

Conclusion
In general, the present study suggested that a better understanding of the migration of cancer cells might allow the development of novel therapeutic concepts. It was proposed that DMH1 regulated lactate excretion in primary tumors and might serve as a novel target for improving therapy. Con ict of Interest The authors declare that they have no competing interests.
Disclosure Summary The authors declare that they have no con ict of interest.
Ehtics approval All the experiments were performed by Using commercially available cell line from ATCC company. Neither human's nor animal's body material was used in this research.
Availability of data and material The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.    b Exposure to 20mM lactate for 24 h had no effect on CT-26 cell activity with or without serum. ** P < 0.01 versus the control. c Lactate stimulated the migration of cancer cells but did not act as a chemoattractant, ** P < 0.01 versus the control. d Lactate speci cally in uenced the migration of cancer cells, but not osmotic effects or metabolic activation. ** P < 0.01 versus the control. Hypoxia, Hypo.
Magni cation, 200×. The results are representative of three independent experiments run in triplicate.
Data represent mean ± SD.