(A) Chemical structure, molecular formula and 308.3495 mol. wt. of LG308. (B) LG308 more potently inhibited the proliferation of several PCa cells than normal prostate epithelium cells. Cells were incubated with different doses of LG308 for 72 hours in 96-well plates, and cell viability was tested by SRB assay (n = 3); **P < 0.01; ***P < 0.001. (C) LG308 inhibited colony formation of PCa cells PC-3M and LNCaP. After treatment by different doses of LG308 in six-well plates for a week, cells were fixed and stained with crystal violet, and the numbers of cell colonies were counted. n = 3; ***P < 0.001. (D) LG308 induced cell-cycle arrest of LNCaP and PC-3M cell lines. Cells were analyzed by flow cytometry after treatment of LG308 with different doses for 24 hours. (E) Effect of LG308 on the expression of G2/M transition related proteins. After incubation with different doses of LG308 for 24 hours, cells were lysed and cdc2, p-cdc2, cdc25c, cyclin B1, and β-actin were measured by Western blotting analysis with their specific antibodies.

(A) LG308 increased the accumulation of mitotic PC-3M cells. PC-3M cells were exposed to different doses of LG308 for 24 hours. Total and mitosis phase cells were counted. The mitotic index was represented by the percentage of mitosis phase cells. Red arrows point to mitotic cells. n = 3; **P < 0.01; ***P < 0.001. (B) LG308 increased the expression of MPM-2 in PC-3M cells. PC-3M cells were treated with different dose of LG308 for 24 hours, and MPM-2 was measured by Western blotting analysis with specific antibodies. (C) LG308 reduced polymerized tubulin in PC-3M and LNCaP cells. Cells were treated with different doses of LG308 or 50 nM colchicine (served as positive control) or 50 nM paclitaxel (served as negative control) for 24 hours. Then separated polymerized tubulin and total tubulin were detected by Western blotting analysis with tubulin antibodies. (D, E) LG308 disrupted the microtubule organization in the interphase and mitotic phase in PC-3M cells. PC-3M cells were treated with different doses of LG308 or 50 nM colchicine (served as positive control) or 50 nM paclitaxel (served as negative control) for 24 hours. Cells were fixed, and the network of microtubulin was stained with α-tubulin antibody (green), and the nuclear DNA was stained by DAPI (blue). Images of interphase cells (D) and mitotic phase cells (E) were taken by confocal microscopy. Red arrows point to microtubulin.

(A, B) LG308 mediated PC-3M cells apoptosis in dose-dependent and time-dependent manner. Cells were treated with different doses of LG308 for 48 hours (A) or with 10 μM LG308 for 24, 48, and 72 hours (B). Then the percentage of apoptosis cells was analyzed using flow cytometry. (C) LG308 destroyed the plasma membrane integrity of PC-3M and LNCaP cells. PC-3M cells were treated with different doses of LG308 for 48 hours and stained by live-dead kit. Representative image at 10 μM was showed. Red represents dead cells; green represents live cells. Red arrows point to dead cells. Quantitative data of different does were shown on the right. n = 3; ***P < 0.001.

(A) PC-3M tumor-bearing mice were treated i.p. with LG308 (25 mg/kg daily) or DMSO (served as control) for 20 days. Tumor volume and mice body weight were measured 3 times a week during LG308 administration, and the weight of the removed tumor was also measured. n = 8; ns, not significant; *P < 0.05; ***P < 0.001. (B) Expression of Ki-67 decreased, whereas MPM-2 increased in the LG308 treatment group. For Ki-67 and MPM-2, IHC staining, sections cut from the paraffin blocks of PC-3M xenograft were carried out using Ki-67 or MPM-2 antibody, and the positive cells (brown) of each group were counted. n = 3; ***P < 0.001. (C) Liver and kidney H&E staining of control and LG308 treatment group.