Selective CDK7 inhibition with BS-181 suppresses cell proliferation and induces cell cycle arrest and apoptosis in gastric cancer

Figures & data

Table 1 In vitro growth inhibitory activity of BS-181 on gastric cancer and normal cell lines

Cell lines	Differentiation	IC50, μM (SD)a
MKN28	Well differentiated	16.8 (4.2)
SGC-7901	Moderately differentiated	19.2 (5.5)
AGS	Poorly differentiated	21.2 (6.1)
BGC823	Undifferentiated	22.4 (5.0)
RGM-1	Normal gastric epithelial cells	6.5 (1.5)

Notes: Different gastric cancer cell lines including MKN28, SGC-7901, AGS, and BGC823 were treated with increasing concentrations of BS-181 for 48 hours. CCK-8 assay showed that gastric cancer cell growth was inhibited by BS-181, with IC₅₀ ranging from 17 μM to 22 μM;

a tested by CCK-8 assay.

Abbreviations: IC₅₀, inhibitory concentration; SD, standard deviation.

Figure 1 BS-181 decreased the migration and invasion ability of BGC823 cells.

Notes: BGC823 cells were treated with BS-181 for 24 hours at indicated concentrations. Migration and invasion assay of BGC823 cells (A). BS-181 significantly decreased the number of migrated (B) and invaded (C) cells. Compared with control group *P<0.05 and **P<0.01; compared to other groups ^##P<0.01.

Figure 2 BS-181 induced cell apoptosis (A) and regulated apoptosis-related protein expressions (B and C).

Notes: BS-181-induced BGC823 cell apoptosis in a dose- and time-dependent manner. Additionally, BS-181 increased expression of proapoptotic proteins and decreased expression of antiapoptotic proteins. Compared with control group *P<0.05 and **P<0.01; compared to other groups ^#P<0.05 and ^##P<0.01.

Figure 3 BS-181-impaired cell cycle progression in BGC823 cells.

Notes: Cell cycle distribution was analyzed by flow cytometry (A). Data are presented as the mean of triplicate experiments (B). Administration of BS-181 significantly increased the percentage of cells in the G0/G1 phase, and significantly decreased the S and G2/M phase fractions.

Table 2 Inhibition of cyclin-dependent protein kinase activity by BS-181

Kinases	Roscovitine IC50, μM (SD)	BS-181 IC50, μM (SD)
CDK7	0.48 (0.12)	0.019 (0.005)
CDK1	1.50 (0.31)	6.70 (2.1)
CDK2	0.09 (0.02)	1.01 (0.32)
CDK4	16.2 (3.50)	27.0 (4.4)
CDK5	0.20 (0.06)	3.60 (0.62)
CDK6	24.5 (5.20)	32.5 (6.7)
CDK9	1.05 (0.34)	4.2 (1.1)

Notes: The mean IC₅₀ values (μM) for roscovitine and BS-181 were obtained from three independent experiments. Roscovitine is a potent and selective CDK inhibitor. Data are expressed as mean ± SD.

Abbreviations: IC₅₀, inhibitory concentration; SD, standard deviation; CDK, cyclin- dependent kinase.

Figure 4 BS-181-inhibited phosphorylation of CDK7 substrates.

Notes: Whole cell lysates were prepared from BGC823 cells treated with BS-181 for 4 hours at indicated concentrations. Immunoblotting was carried out using antibodies for RNA polymerase II or Pol II phosphorylated at Ser5 in the C-terminal domain (A). Data are presented as mean ± SD of three independent experiments (B). Compared to other groups ^#P<0.05 and ^##P<0.01.

Figure 5 BS-181-inhibited gastric cancer growth in vivo and increased survival rate.

Notes: The change in tumor volume (A) was determined for each animal, as tumor volume relative to the tumor volume of each animal at day 1; mice body weights (B) were similar between groups for a 14-day observation; treatment with BS-181 significantly increased survival rate in an additional 30-day observation (C); roscovitine was used as a positive control. Compared to control group *P<0.05, **P<0.01, and ***P<0.001. Compared to other groups ^#P<0.05.