Fig 1.
Structure of CA4 and related anti cancer coumpounds.
Fig 2.
CA4 and biphenyl analogues inhibit proliferation of cancer cells.
Various cancerous and non-cancerous cells were treated with the indicated drug at the indicated dose for 48 hours and the WST-1 reagent was used to quantify proliferation. Absorbance was read at 450 nm and expressed as a % of DMSO control. MG-63 = osteocarcoma cells; HCT-116, HT-29 = colorectal cancer; HOb = normal human osteoblasts. Values are expressed as mean ± SD from quadruplicates of at least three independent experiments.
Table 1.
IC50 values of compounds MP5-F9, MP5-G9, and CA4 in various cancerous cell lines.
Fig 3.
CA4 and biphenyl analogues induce apoptosis in cancer cells.
Various cancerous and non-cancerous cells were treated with the indicated drug at the indicated dose for 48 hours. Cells were then probed with annexin V (green fluorescent probe that binds to apoptotic cells) and propidium iodide (PI), a red fluorescent probe that binds to cells with permeabalized membranes. Percentage of annexin V and PI positive cells was quantified using image-based cytometry. Blue bar represents viable cells; orange bar represents PI and annexin V positive cells; green bar represents annexin V positive cells; red bar represents PI positive cells. Staurosporine (STS) was utilized as a positive control. Values are expressed as mean ± SD from at least three independent experiments. * p < 0.05 vs. DMSO control.
Fig 4.
CA4 and biphenyl analogues reduce growth and proliferation of colorectal cancer spheroids selectively.
(A) HCT116 cancer cells and (B) NCM460 normal colon mucosa cells were grown in ultra-low adherent round-bottomed 96-well plates for 48 hours to establish spheroid formation. Subsequently, spheroids were treated with indicated dose of either CA4 or biphenyl derivative for 72 hours. Spheroid morphology and size was monitored via differential interference contrast microscopy. Scale bar = 500 microns. (C) The WST-1 reagent was used to quantify proliferation. Absorbance was read at 450 nm and expressed as a % of DMSO control. * p < 0.05.
Fig 5.
CA4 and biphenyl analogues induced G2/M arrest precedes mitochondrial collapse and apoptotic induction.
(A) Treated cells (dose and time are indicated) were fixed overnight with ice cold 80% ethanol and stained with propidium iodide. Cell cycle profiles were generated using image-based cytometry. Values are expressed as mean ± SD of three independent experiments. * p < 0.05 versus DMSO control; *** p < 0.001 versus DMSO control; **** p < 0.0001 versus DMSO control. (B) Treated cells were probed with tetramethylrhodamine methyl ester (TMRM). Taxol and pancratistatin (PST) treated cells served as positive controls. Percentage of TMRM positive cells was quantified using image-based cytometry. Values are expressed as mean ± SD of three independent experiments. * p < 0.05 versus DMSO control; **** p < 0.0001 versus DMSO control. (C) Treated cells were probed with annexin V and propidium iodide (PI). Percentage of annexin V and PI positive cells was quantified using image-based cytometry. 1.0 μM staurosporine (STS) treated cells served as a positive control. Blue bar represents viable cells; orange bar represents PI and annexin V positive cells; green bar represents annexin V positive cells; red bar represents PI positive cells. Values are expressed as mean ± SD from three independent experiments. * p < 0.05 vs. DMSO control.
Fig 6.
Small molecule inhibitors of mitotic arrest able to ameliorate mitotic arrest, mitochondrial collapse, and apoptotic induction.
MV-4-11 cells were treated with either CA4, MP5-F9, MP5-G9, or nocodazole (NOC), alone or co-incubated with either reversine (rev) or RO-3306, for 24 hours. (A) Whole cell lysates were electrophoresed, transferred to a PVDF membrane and probed for cyclin B1 and β-actin. Bands were visualized with enhanced chemiluminescence reagent and densitometry analyses were performed using ImageJ software. Values are expressed as the ratio cyclin B1 band intensity over β-actin band intensity and are normalized to DMSO control. Western blot images are representative of three independent trials. (B) Treated cells were probed with tetramethylrhodamine methyl ester (TMRM). Percentage of TMRM positive cells was quantified using image-based cytometry. Values are expressed as mean ± SD of at least three independent experiments. ** p < 0.01; *** p < 0.001. (C) Treated cells were probed with green fluorescent annexin V and propidium iodide (PI), a red fluorescent probe. Percentage of annexin V and PI positive cells was quantified using image-based cytometry. Blue bar represents viable cells; orange bar represents PI and annexin V positive cells; green bar represents annexin V positive cells; red bar represents PI positive cells. Values are expressed as mean ± SD from at least three independent experiments. *** p < 0.001; **** p < 0.0001.
Fig 7.
Cells with defective extrinsic pathway of apoptosis experience delayed response to treatment with CA4 and biphenyl analogues.
(A) Wild-type E6-1 leukemia cells and dominant-negative Fas-associated via death domain (DN FADD) leukemia cells were treated with the indicated drug at the indicated dose for 48 hours and the WST-1 reagent was used to quantify proliferation. Absorbance was read at 450 nm and expressed as a % of DMSO control. (B, C) Cells were treated for either (B) 24 hours or (C) 48 hours and were probed with annexin V and propidium iodide (PI). Percentage of annexin V and PI positive cells was quantified using image-based cytometry. Blue bar represents viable cells; orange bar represents PI and annexin V positive cells; green bar represents annexin V positive cells; red bar represents PI positive cells. Values are expressed as mean ± SD from three independent experiments. * p < 0.05 vs. DMSO control; **** p < 0.0001 vs. DMSO control.
Fig 8.
Predicted Tubulin Affinity Correlates with Cytotoxicity of CA4 and Biphenyl Analogues.
Green = hydrophobic residues; red = polar residues; white = non-interacting residues. Using Molecular Operating Environment (MOE) software, CA4, MP5-F9, and MP5-G9 were separately docked in the colchicine binding pocket, whose crystal structure was downloaded from the protein data bank (PDB code: 1SA0). Red arrow indicates the same hydrophobic pocket within the overall colchicine binding site, which is occupied by the ethylene linker of CA4. (A) CA4 (B) MP5-G9 (C) MP5-F9 (D) Merged image of CA4 and CA4 analogues docked to colchicine binding site. Yellow = cis-CA4; Green = MP5-G9; Blue = MP5-F9.
Table 2.
CA4 and biphenyl analogues are predicted to bind favourably to the colchicine binding site of tubulin.