naphthoquinones and Esophageal-Squamous-Cell-Carcinoma

Lapachol is a 1,4-naphthoquinone that is isolated from the Bignoniaceae family. It has been reported to exert anti-inflammatory, antibacterial, and anticancer activities. However, the anticancer activity of lapachol and its molecular mechanisms against esophageal squamous cell carcinoma (ESCC) cells have not been fully investigated. Herein, we report that lapachol is a novel ribosomal protein S6 kinase 2 (RSK2) inhibitor that suppresses growth and induces intrinsic apoptosis in ESCC cells. We found that lapachol strongly attenuates downstream signaling molecules of RSK2 in ESCC cells and also directly inhibits RSK2 activity in vitro. The RSK protein is highly activated in ESCC cells and knockdown of RSK2 significantly suppresses anchorage-dependent and anchorage-independent growth of ESCC cells. Additionally, lapachol inhibits anchorage-dependent and anchorage-independent growth of ESCC cells, and the inhibition of cell growth by lapachol is dependent on the expression of RSK2. We also found that lapachol induces mitochondria-mediated cellular apoptosis by activating caspases-3, -7, and PARP, inducing the expression of cytochrome c and BAX by inhibiting downstream molecules of RSK2. Overall, lapachol is a potent RSK2 inhibitor that might be used for chemotherapy against ESCC.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Cell Line, Tumor; Cell Proliferation; Esophageal Squamous Cell Carcinoma; Humans; Naphthoquinones; Ribosomal Protein S6 Kinases, 90-kDa; Signal Transduction

Esophageal squamous cell carcinoma (ESCC) is one of the deadliest cancers, and it requires novel treatment approaches and effective drugs. In the present study, we found that treatment with plumbagin, a natural compound, reduced proliferation and survival of the KYSE150 and KYSE450 ESCC cell lines in a dose-dependent manner in vitro. The drug also effectively inhibited the viability of primary ESCC cells from fresh biopsy specimens. Furthermore, plumbagin-induced mitotic arrest and massive apoptosis in ESCC cells. Notably, the drug significantly suppressed the colony formation capacity of ESCC cells in vitro and the growth of KYSE150 xenograft tumors in vivo. At the molecular level, we found that exposure to plumbagin decreased both polo-like kinase 1 (PLK1) and phosphorylated protein kinase B (p-AKT) expression in both ESCC cell lines. Enforced PLK1 expression in ESCC cells not only markedly rescued cells from plumbagin-induced apoptosis and proliferation inhibition but also restored the impaired AKT activity. Furthermore, signal transducer and activator of transcription 3 (STAT3), a transcription factor of PLK1, was also inactivated in plumbagin-treated ESCC cells; however, the overexpression of a constitutively activated STAT3 mutant, STAT3C, reinstated the plumbagin-elicited blockade of PLK1-AKT signaling in ESCC cells. Taken together, these findings indicate that plumbagin inhibits proliferation and potentiates apoptosis in human ESCC cells in vitro and in vivo. Plumbagin may exert these antitumor effects by abrogating STAT3-PLK1-AKT signaling, which suggests that plumbagin may be a novel, promising anticancer agent for the treatment of ESCC.

Topics: Animals; Apoptosis; Carcinogenesis; Cell Cycle Checkpoints; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Cell Survival; Down-Regulation; Esophageal Neoplasms; Esophageal Squamous Cell Carcinoma; Female; Humans; Mice, Nude; Naphthoquinones; Polo-Like Kinase 1; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Signal Transduction; STAT3 Transcription Factor; Xenograft Model Antitumor Assays

Although plumbagin, a natural naphthoquinone, has exhibited antiproliferative activity in numerous types of cancer, its anticancer potential in esophageal squamous cell carcinoma (ESCC) remains unclear. In the present study, the effect of plumbagin on the growth of ESCC cells was investigated in vitro and in vivo. ESCC cells were treated with plumbagin and tested for cell cycle distribution and apoptosis. The involvement of STAT3 signaling in the effect of plumbagin was examined. The results demonstrated that plumbagin treatment suppressed ESCC cell viability and proliferation, yet normal esophageal epithelial cell viability was not affected. Plumbagin treatment increased the proportion of cells in the G0/G1 phase of the cell cycle and decreased the proportion of cells in the S phase. Furthermore, plumbagin‑treated ESCC cells displayed a significantly greater % of apoptotic cells. Western blot analysis confirmed that plumbagin upregulated tumor protein p53 and cyclin‑dependent kinase inhibitor 1A (also known as p21), while it downregulated cyclin D1, cyclin‑dependent kinase 4, and induced myeloid leukemia cell differentiation protein Mcl‑1. Mechanistically, plumbagin inhibited STAT3 activation, and overexpression of constitutively active STAT3 reversed the plumbagin‑mediated growth suppression in ESCC cells. In vivo studies demonstrated that plumbagin delayed the growth of ESCC xenograft tumors and reduced STAT3 phosphorylation. Overall, plumbagin was demonstrated to target STAT3 signaling and to inhibit the growth of ESCC cells both in vitro and in vivo, suggesting that it may represent a potential anticancer agent for ESCC.

Topics: Animals; Apoptosis; Carcinoma, Squamous Cell; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cell Survival; Esophageal Neoplasms; Esophageal Squamous Cell Carcinoma; G1 Phase; Humans; Mice; Naphthoquinones; Resting Phase, Cell Cycle; STAT3 Transcription Factor; Xenograft Model Antitumor Assays

Survivin and transcription factor p65 (NF‑κB p65) participate in the progression of esophageal squamous cell carcinoma (ESCC). However, the mechanism of NF‑κB p65 activation in ESCC remains to be elucidated. The aim of the present study was to investigate the role of survivin in the activation of NF‑κB p65 in ESCC. The expression levels of survivin, NF‑κB p65, inhibitor of nuclear factor κB kinase subunit α (IKKα) and inhibitor of nuclear factor κB kinase subunit β (IKKβ) were detected in ESCC tissue samples. Eca109 and KYSE150 cells were cultured and survivin activity was modulated via transfection with an overexpression plasmid, a small hairpin RNA plasmid and a specific inhibitor. Quantitative reverse transcription-polymerase chain reaction and western blotting assays were conducted to assess the effects of survivin on the expression levels of IKKα, IKKβ and NF‑κB p65. Cell cycle and apoptosis assays were conducted to detect surviving-dependent cellular behavior changes. In addition, the luciferase reporter gene assay and chromatin immunoprecipitation assay were conducted to determine the genomic sites responsible for surviving-induced activation of NF‑κB p65. The present study demonstrated that the expression of survivin is positively correlated with IKKα and IKKβ in ESCC tissues. Survivin affected the mRNA and protein expression levels of IKKα, IKKβ, and NF‑κB p65 in Eca109 and KYSE150 cells. Furthermore, survivin increased the transcriptional activity of the IKKβ promoter and bound to the IKKβ promoter region in the Eca109 cells. Downregulation of survivin arrested the cell cycle at the G2/M phase and induced apoptosis. Results of the present study suggest that survivin activates NF‑κB p65 in Eca109 cells via binding to the IKKβ promoter region and upregulating IKKβ promoter transcriptional activity. Survivin overexpression activates NF‑κB p65, which is important in the acquisition and maintenance of the oncogenic characteristics of ESCC.

Topics: Apoptosis; Carcinoma, Squamous Cell; Cell Count; Cell Line, Tumor; Cell Survival; Chromatin Immunoprecipitation; Down-Regulation; Esophageal Neoplasms; Esophageal Squamous Cell Carcinoma; G2 Phase Cell Cycle Checkpoints; Gene Knockdown Techniques; Humans; I-kappa B Kinase; Imidazoles; Inhibitor of Apoptosis Proteins; Naphthoquinones; Promoter Regions, Genetic; Protein Binding; RNA, Small Interfering; Survivin; Transcription Factor RelA; Up-Regulation

Survivin is overexpressed in cancer cells and plays a crucial role in apoptosis evasion. YM155, a small-molecule inhibitor of survivin, could enhance the cytotoxicity of various DNA-damaging agents. Here, we evaluated the radiosensitizaion potential of YM155 in human esophageal squamous cell carcinoma (ESCC).. Cell viability was determined by CCK8 assay. The radiosensitization effect of YM155 was evaluated by clonogenic survival and progression of tumor xenograft. Cell cycle progression was determined by flow cytometric analysis. Radiation-induced DNA double strand break (DSB) and homologous recombination repair (HRR) were detected by the staining of γ-H2AX and RAD51, respectively. Expression of survivin and cell cycle regulators was detected by Western blot analysis.. YM155 induced radiosensitization in ESCC cell lines Eca109 and TE13, associated with the abrogation of radiation induced G2/M checkpoint, impaired Rad51 focus formation, and the prolongation of γ-H2AX signaling. G2/M transition markers, including the activation of cyclinB1/Cdc2 kinase and the suppression of Cdc2 Thr14/Tyr15 phosphorylation were induced by YM155 in irradiated cells. The combination of YM155 plus irradiation delayed the growth of ESCC tumor xenografts to a greater extent compared with either treatment modality alone.. Our findings suggest that the abrogation of G2 checkpoint and the inhibition of HRR contribute to radiosensitization by YM155 in ESCC cells.

Topics: Animals; Antineoplastic Agents; Blotting, Western; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Survival; Esophageal Neoplasms; Esophageal Squamous Cell Carcinoma; Flow Cytometry; Fluorescent Antibody Technique; G2 Phase Cell Cycle Checkpoints; Humans; Imidazoles; Inhibitor of Apoptosis Proteins; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Naphthoquinones; Radiation-Sensitizing Agents; Recombinational DNA Repair; Survivin; Xenograft Model Antitumor Assays