The Anti-Proliferative Activity of the Hybrid TMS-TMF-4f Compound Against Human Cervical Cancer Involves Apoptosis Mediated by STAT3 Inactivation

Abstract

We previously reported the potential anti-proliferative activity of 3-(5,6,7-trimethoxy-4-oxo-4H-chromen-2-yl)-N-(3,4,5-trimethoxyphenyl) benzamide (TMS-TMF-4f) against human cancer cells; however, the underlying molecular mechanisms have not been investigated. In the present study, TMS-TMF-4f showed the highest cytotoxicity in human cervical cancer cells (HeLa and CaSki) and low cytotoxicity in normal ovarian epithelial cells. Annexin V-FITC and propidium iodide (PI) double staining revealed that TMS-TMF-4f-induced cytotoxicity was caused by the induction of apoptosis in both HeLa and CaSki cervical cancer cells. The compound TMS-TMF-4f enhanced the activation of caspase-3, caspase-8, and caspase-9 and regulated Bcl-2 family proteins, which led to mitochondrial membrane potential (MMP) loss and resulted in the release of cytochrome c and Smac/DIABLO into the cytosol. Also, TMS-TMF-4f suppressed both constitutive and IL-6-inducible levels of phosphorylated STAT3 (p-STAT3) and associated proteins such as Mcl-1, cyclin D1, survivin, and c-Myc in both cervical cancer cells. STAT-3 overexpression completely ameliorated TMS-TMF-4f-induced apoptotic cell death and PARP cleavage. Docking analysis revealed that TMS-TMF-4f could bind to unphosphorylated STAT3 and inhibit its interconversion to the activated form. Notably, intraperitoneal administration of TMS-TMF-4f (5, 10, or 20 mg/kg) decreased tumor growth in a xenograft cervical cancer mouse model, demonstrated by the increase in TUNEL staining and PARP cleavage and the reduction in p-STAT3, Mcl-1, cyclin D1, survivin, and c-Myc expression levels in tumor tissues. Taken together, our results suggest that TMS-TMF-4f may potentially inhibit human cervical tumor growth through the induction of apoptosis via STAT3 suppression.

Role of 2′-Hydroxycinnamaldehyde In The Induction of Apoptosis Via A Reactive Oxygen Species-Dependent JNK Pathway in Human Promyelocytic HL-60 Leukemia Cells

“…After CYP injection, HL-60 cells (1 × 10 6 per site) were inoculated subcutaneously into the right side of the ank of male BALB/c nude mice. Tumor size was checked with a caliper [24] once per 3 days in a week and calculated as V = π/6 × (length) × (width) 2 .…”

KCP10043F Represses the Proliferation of Human Non-Small Cell Lung Cancer Cells by Caspase-Mediated Apoptosis via STAT3 Inactivation

“…The 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assay was used as previously described to examine cytotoxicity [23]. briefly, cells were seeded in a 96-well plate, and each well contains 5 × 10 4 cells/mL in 100 µL of the medium.…”

“…After co-treatment with KCP10043F and interleukin-6 (IL-6), cells were washed with PBS and then were fixed with 4% paraformaldehyde in PBS at 4 • C. The next day, to induce cell permeability, cells were washed with PBS three times and were incubated with 0.3% Triton-X 100/PBS for 1 h at 25 • C as described previously [23]. counterstaining with DAPI.…”

“…The male Bagg Albino (BALB)/c nude mice (7-week-old, 20-23 g) were obtained from Nara Biotec Co. (Pyeongtaek, Republic of Korea). All animal experiments were approved by the Committee for the Care and Use of Laboratory Animals in the Kyung Hee University (KHUASP(SE)-18-146) and were performed according to the National Institutes of Health (NIH) and Kyung Hee University Guidelines for Laboratory Animals Care and Use as described previously [23]. The animal protocol was designed to minimize pain and discomfort to the animals.…”

“…To establish the A549 xenograft model, subcutaneous implantation of A549 cells was performed as previously described [23]. Tumor size was checked with a caliper three times per week and calculated as V = π/6 × (length) × (width) 2 [24].…”