In order to investigate the effect of GBK on the migration and invasion of breast cancer cells, would healing and transwell cell invasion assays were performed. The triple negative breast cancer cells SUM-159, ER-positive breast cancer cells MCF-7 and normal human epithelial mammary cell line MCF-10A were scratched and photographed, and different concentrations of GBK were applied. After 24 hours of culture, cell images were captured, and the cell motility changes were analyzed. In order to rule out the influence of cell proliferation, we switched to 1% low serum medium after the scratch treatment and took pictures within one cell cycle.
The results showed that the wound closure ability of both SUM-159 and MCF-7 cells was significantly inhibited by GBK. The inhibition rate of cell migration is 59% in 150 µg/mL (IC50 of SUM-159) concentration of GBK treatment group, and further increased to 71% in the 300 µg/mL treatment group (Fig. 1A and B). Moreover, in SUM-159 cells GBK treatment inhibited invasion in transwell invasion assay by 64 % in the 150 µg/mL concentration and 92 % in the 300 µg/mL concentration, respectively (Fig. 1C and D). In contrast, apply of GBK in MCF-7 human non-invasive breast cancer cells also impaired cell invasion, but in a less obvious level (Fig. 1C and D). Taken together, these data suggest an anti-migration and anti-invasion role of GBK in breast cancer cells, especially in invasive breast cancer cells.
Considering that some miRNAs could function as tumor suppressor and has been implicated in directly inhibiting cancer progression, they may act as specific drug target in cancer treatment. For instance, miR-21 is involved in the regulation of apoptosis in breast cancer cells [7], miR-151 is found to affect the development of breast cancer through the action of DNA repair process, and miR-421 can inhibit the migration and invasion of breast cancer by targeting MTA1[8, 9].We selected nine miRNAs closely related to the occurrence and development of breast cancer based on the former research. Among the nine miRNAs selected, miR-22, miR-31, miR-411 and miR-421 function as tumor suppressor, while miR-21, miR-145, miR-150, miR-182 and miR-217 could promote cancer development[10–20].
In order to determine the potential miRNA target of GBK, RT-qPCR was used to detect the expression of those miRNA in mouse serum from breast cancer xenograft mouse model after GBK treatment (Supplementary Fig. 1). However, the results showed that after treatment of GBK, the expression level of nice miRNAs in serum was slightly higher than those in control group. Although there are some changes between the experimental group and the control group, the results are not statistic significant. We then performed the experiments in breast cancer cell lines MCF-7 and SUM-159 to investigate the function of GBK on the expression of breast cancer related miRNAs. Different concentration of GBK was added to the cells for 48h and miRNA expression was detection by RT-qPCR. The expression of miR-31 was consistently upregulated after GBK treatment in two breast cancer cell lines tested (Fig. 2B). The expression of other miRNAs to GBK treatment was not consistent and thus excluded for the further investigation (Fig. 2A-I).
By analyzing the expression of nine breast cancer related miRNAs, it is preliminarily indicated that the tumor suppressor effect of GBK may be closely related to miR-31.
The expression level of miR-31 target genes related to cell migration is down-regulated after GBK treatment
In previous studies, the function of miR-31 has been shown to be highly related with the progression and metastasis of breast cancer, and miR-31 exerts a fundamental role in regulating the invasion-metastasis cascade via targets genes critical in these processes, such as cytoskeletal rearrangement proteins and pivotal genes in cancer-associated fibroblasts (CAFs). RhoA, which participates in the regulation of actin cytoskeleton, was shown to be a direct target of miR-31[21, 22]. Moreover, another member of the Rho family, RHOBTB1, was shown to be a target of miR-31 in colon cancer[23]. WAVE3, an actin remodeling protein, was shown to be overexpressed in invasive breast cancer cells due to miR-31 down-regulation, and its expression promoted cancer cell migration and invasion[24]. Homeobox gene SATB2 was shown to be a direct target of miR-31 in CAF and involved in increasing tumor cell migration and invasion[25].
We screened nine target genes closely related to the anti-metastatic function of miR-31, monitoring their expression in SUM-159 cells after GBK treatment. The expression of RHOBTB1, RhoA, SATB2, and WAVE3 are all down-regulated after 48 hours of GBK treatment, indicating that the tumor suppressor effect of GBK may be related to invasion-metastasis related signaling pathways (Fig. 3A-I).
In order to further validate the effect of GBK on the expression of invasion-metastatic genes, western blot analysis was performed. It was demonstrated that the expression level of RhoA, WAVE3 and SATB2 was all reduced after GBK treatment (Fig. 3J). Thus, it is speculated that GBK inhibits the migration and invasion of breast cancer cells by promoting the expression of miR-31, in turn inhibiting the expression of miR-31 target genes RhoA, WAVE3 and SATB2.
Expression of miR-31 and its host gene lncRNA LOC554202 is upregulated by inhibiting promoter hypermethylation after GBK treatment
It was pointed out that miR-31 locates in the intronic sequence of long non-coding RNA (lncRNA) LOC554202, and its transcriptional activity is regulated by LOC554202 (Fig. 4A) [24]. It was also demonstrated that the major mechanisms for silencing miR-31 in triple-negative breast cancer (TNBC) is hypermethylation of the CpG island of the LOC554202 promoter region, which may become a new entry point for TNBC treatment [24].
We tested the expression of LOC554202 in ER-positive breast cancer cell lines MCF-7 and TNBC cell line SUM-159, and found that the expression of LOC554202 was up-regulated under GBK treatment (Fig. 4B and C). Then we selected two CpG sites in LOC554202 and methylation-specific PCR (MSP) technology was used to detect changes of the methylation level of these sites after GBK treatment. We found the methylation levels at CpG MSP Set2 decreased significantly under GBK treatment in a dose-dependent manner in two cell lines (Fig. 4D and E). These data indicate that GBK treatment affects the epigenetic modification at CpG sites and plays an important role in upregulating of both the LOC554202 and miR-31 genes.
Combination treatment with GBK and DDP/VP-16 has synergistic and dose reduction potential in the proliferation of breast cancer cells
The chemotherapy drugs cisplatin (DDP) and Vepeside (VP-16) are widely used as first-line therapy for metastatic breast cancer and small cell lung cancer[26, 27]. In the precious study, we demonstrated that GBK has specific inhibitory effect on breast cancer cells compared with normal breast cells and other types of cancer cells [28]. In this preclinical study, we want to evaluate the effect of GBK in combination with DDP/VP-16 as agents in breast cancer cells. The sensitivity of SUM-159 and MCF-7 cell lines to growth inhibition during 48 hours exposure to GBK and DDP/VP-16 was determined as single agents, and in combination at 6 concentrations between 0.1× and 4× their respective IC50 concentrations for DDP, as well as 0.2× and 4× their respective IC50 concentrations for VP-16. The effect of combined treatment was cell type dependent. The combination of DDP/VP-16 with GBK at all concentrations led to greater growth inhibition compared to either agent alone for the SUM-159 (Fig. 5A-E) and MCF-7 (Fig. 5F-J) cell lines, although the increase was smaller for the MCF-7 cells.
These results demonstrated that chemotherapy drugs DDP/VP-16 combination with GBK has synergistic and dose reduction potential in the proliferation of breast cancer cells SUM-159 and MCF-7, indicating a potential guiding significance for clinical combination treatment.
Combination treatment with GBK and DDP impaired the independent viability of breast cancer cells
It has been showed that GBK treatment led to an upregulation of the expression of miR-31 and its host gene lncRNA LOC554202 in breast cancer cells when acting alone. In order to study whether the combination medication treatment with GBK and DDP can also have the same synergistic function, RT-qPCR was performed to detect the target genes expression of miR-31 in SUM-159 and MCF-7 after drug treatment. It was revealed that when the cells were treated with GBK combined with DDP, the expression of SATB2 and RhoA was decreased significantly in SUM-159 cells, and there were no significant changes in expression levels of other genes (Fig. 7). It was also indicated that combination treatment has an obvious synergistic effect in SUM-159 cells than that in MCF-7 cells, meanwhile, the low concentration of GBK combined with DDP appears have better synergistic effects than high concentration counterpart (Fig. 7).
Combination treatment with GBK and DDP has synergistic and dose dependent effect on the inhibition of tumor growth in vivo
To further analyze whether the combination treatment with GBK and DDP has a clear inhibitory effect on breast cancer progression in vivo, NOD/SCID immunodeficient mice were used to construct a human breast cancer cell xenograft model by subcutaneously inoculated with SUM-159 cells. All of the mice developed subcutaneous xenografts, and then were divided into four groups and administered intraperitoneally with GBK 10 mg/(kg.1D) and DDP 1 mg/(kg.2Ds) in Group A, GBK 5 mg/(kg.1D) and DDP 1 mg/(kg.2Ds) in Group B, 0.9% saline in group C, and DDP 1 mg/(kg.2Ds) in Group D when the tumor volume reached 8 mm3. During 24 days of continuous injection, the notable anti-tumor effects were observed.
We found that DDP alone in group D could inhibit tumor proliferation, and under the combined treatment of GBK and DDP, the tumor volume and average tumor weight of mice in group A and B were further reduced than those in D, indicating that combination treatment with drugs of DDP and GBK has synergistic and dose dependent effect on the inhibition of breast cancer cell proliferation in vivo (Fig. 8A-C).
In addition, the body weight of mice in group C which were administered with saline increased slightly, while the body weight of mice in D that only administered with DDP decreased slightly. Interestingly the body weight of mice in group A and B decreased obviously with the combination treatment with DDP and GBK, and the weight loss of group A was larger than that of B (Fig. 8D). It was indicated that GBK could also perform as an antihyperlipidemic drug alone with its anti-tumor properties.