Investigation and development of natural products that target chemotherapy resistance factors in cancer cells

Abstract

Heat shock proteins (HSPs) play an important role in several tumors; contribute to anti-cancer drug resistance, cell proliferation, and metastasis; and have been suggested as a major cause of failed anti-cancer drug treatment. In addition, cancer stem cells (CSCs) have been identified in many types of malignancies, including leukemia, breast, colorectal, and brain cancers, and are a leading cause of failed cancer treatment owing to their anti-cancer drug and radiation therapy resistance. Therefore, many researchers, including our group, have investigated and developed natural products that target chemotherapy resistance factors in cancer cells. This review introduces the inhibitors of chemotherapy resistance factors discovered using a unique assay system.

Keywords: Cancer stem cell; Chemotherapy resistance factor; Heat shock protein; Wnt/β-catenin pathway.

Fig. 1

Effects of Hypericum erectum constituents on cell proliferation, cell death, and heat shock protein (HSP) expression in HeLa cells. A Chemical structures of constituents (1a, 1b, 2a, and 2b) isolated from H. erectum. B HeLa cells were treated for 24 h with 50 μM of the indicated compounds, ADR (0.5 and 1.0 μg/ml), or a combination of both. The number of cells that entered mitosis and the number of dead cells were counted by time-lapse imaging. The results are reported as mean ± standard deviation of triplicate determinations. Statistical significance was analyzed using the Tukey–Kramer test (*P < 0.01 and **P < 0.001 compared with 1.0 μg/ml ADR-treated cells). C Western blots of HSP105, 90, and 70 expression levels in HeLa cells treated with 1a and 1b for 24 h. (D) Bar graphs of the relative expression levels of HSPs/β-actin compared to those of control in the western blots using ImageJ software

Fig. 2

Effects of compounds produced by Penicillium maximae JKYM-AK1 on heat shock protein promoter activity in pGL105/C3H cells and on cell proliferation and death in HeLa cells. A Chemical structures of compounds (3–9) produced by P. maximae JKYM-AK1. B Inhibitory effects of 3–9 on heat shock protein promoter activity as determined using a luciferase assay system with pGL105/C3H cells. The percentages of luciferase activities and cell viabilities are described as means ± SD (n = 3) from three independent experiments. Statistical significance was analyzed using Dunnett’s test (*P < 0.01, ^#P < 0.01 compared with each control group). C Effects of 3–9 on cell proliferation and death. The numbers of mitotic entry and dead cells were counted during time-lapse imaging. The percentages of mitotic entry and dead cells are reported as means ± SD of three different visual fields. More than 100 cells were captured in each field. Statistical significance was analyzed using the Tukey–Kramer test [*P < 0.01 compared with the control group, **P < 0.01 compared with the ADR (1.0 mg/ml) group, ***P < 0.01 compared with the ADR (2.0 mg/ml) group.]

Fig. 3

The role of Wnt/β-catenin pathway on CSCs, synthesis of 10, 10da, and related compounds together with the expression levels of mediator and target genes of the Wnt/β-catenin signaling pathway in HT-29 cells treated with 10da. A In the absence of Wnt signaling, serine phosphorylation of β-catenin by the Axin–glycogen synthase kinase–3β complex targets β-catenin for ubiquitination degradation. In the presence of Wnt signaling, the activity of the Axin complex is inhibited and β-catenin enter the nucleus. Then, β-catenin binds to TCF to form a complex, which enhances gene expression that related to pluripotency of CSCs. B Synthesis of linderapyrone (10) and related compounds for the structure–activity relationship study. C HT-29 cells were treated with the indicated concentrations of the compounds for 24 h. Cell lysates were collected, and the Wnt/β-catenin signaling-related proteins were detected by western blotting. D HT-29 cells were treated with the indicated concentrations of compounds for 24 h. Quantitative reverse transcription PCR (RT-qPCR) was performed to evaluate c-myc and survivin gene expression levels. The amount of β-actin mRNA was used to normalize the data. Data are presented as mean ± SD of three independent experiments (*P < 0.05, **P < 0.01, compared with the control)

Fig. 4

Identification of the target proteins of 10a. A Scheme for the fixation of compounds 11 and 12 onto magnetic FG beads with epoxy linkers. B Binding proteins for 11a and 12a were purified from HT-29 cell extracts. The purified proteins were subjected to SDS-PAGE, followed by silver staining (B) or western blotting C. The input lane represents the 5% HT-29 cell extract used for the binding assay. ^※We could not identify this protein in this study. (D, F, and G) HT-29 cells were treated with 10a at the indicated concentrations. After incubation for 23 h, the cells were treated with ( +) or without ( −) TGF-β (10 ng/mL) in the presence of 10a for 1 h. Importin7, Smad2, and Smad3 proteins were detected by western blotting. (G) Cells were fixed and stained with an anti-importin7 antibody. Nuclei were stained with Hoechst33342. In all Figures, scale bar = 10 mm

Fig. 5

Knockdown of importin7 downregulates the expression of the target genes of the Wnt/β-catenin signaling pathway. A, B Non-target siRNA (siControl) and two independent importin7 siRNA (siIPO7-1 and siIPO7-2) were transfected into HT-29 cells. A Importin7 protein was detected using western blotting. The relative expression levels of importin7/β-actin compared to those of the control were shown. B RT-qPCR was performed to determine the mRNA levels for c-Myc and Survivin genes. The level of β-actin mRNA expression was used to normalize the data. The data are presented as mean ± SD of three independent experiments (**P < 0.01 compared with siControl). C A model of the crosstalk between the Wnt/β-catenin pathway and the Smad pathway together with the role of importin7