3-Deoxysappanchalcone Inhibits Skin Cancer Proliferation by Regulating T-Lymphokine-Activated Killer Cell-Originated Protein Kinase in vitro and in vivo

Abstract

Background: Skin cancer is one of the most commonly diagnosed cancers worldwide. The 5-year survival rate of the most aggressive late-stage skin cancer ranges between 20 and 30%. Thus, the discovery and investigation of novel target therapeutic agents that can effectively treat skin cancer is of the utmost importance. The T-lymphokine-activated killer cell-originated protein kinase (TOPK), which belongs to the serine-threonine kinase class of the mitogen-activated protein kinase kinase (MAPKK) family, is highly expressed and activated in skin cancer. The present study investigates the role of 3-deoxysappanchalcone (3-DSC), a plant-derived functional TOPK inhibitor, in suppressing skin cancer cell growth.

Purpose: In the context of skin cancer prevention and therapy, we clarify the effect and mechanism of 3-DSC on different types of skin cancer and solar-simulated light (SSL)-induced skin hyperplasia.

Methods: In an in vitro study, western blotting and in vitro kinase assays were utilized to determine the protein expression of TOPK and its activity, respectively. Pull-down assay with 3-DSC and TOPK (wild-type and T42A/N172 mutation) was performed to confirm the direct interaction between T42A/N172 amino acid sites of TOPK and 3-DSC. Cell proliferation and anchorage-independent cell growth assays were utilized to determine the effect of 3-DSC on cell growth. In an in vivo study, the thickness of skin and tumor size were measured in the acute SSL-induced inflammation mouse model or SK-MEL-2 cell-derived xenografts mouse model treated with 3-DSC. Immunohistochemistry analysis of tumors isolated from SK-MEL-2 cell-derived xenografts was performed to determine whether cell-based results observed upon 3-DSC treatment could be recapitulated in vivo.

Results: 3-DSC is able to inhibit cell proliferation in skin cancer cells in an anchorage-dependent and anchorage-independent manner by regulation of TOPK and its related signaling pathway in vitro. We also found that application of 3-DSC reduced acute SSL-induced murine skin hyperplasia. Additionally, we observed that 3-DSC decreased SK-MEL-2 cell-derived xenograft tumor growth through attenuating phosphorylation of TOPK and its downstream effectors including ERK, RSK, and c-Jun.

Conclusions: Our results suggest that 3-DSC may function in a chemopreventive and chemotherapeutic capacity by protecting against UV-induced skin hyperplasia and inhibiting tumor cell growth by attenuating TOPK signaling, respectively.

Keywords: 3-deoxysappanchalcone; T-LAK cell-originated protein kinase; cancer growth; skin cancer; skin hyperplasia; solar sinulated light.

FIGURE 1

3-Deoxysappanchalcone (3-DSC) inhibits T-LAK cell-originated protein kinase (TOPK) kinase activity by binding to Thr42 and Asn172. (A) The protein expression levels of TOPK and its downstream protein effectors in Normal Human Dermal Fibroblast (NHDF) and skin cancer cell lines (A431, SK-MEL2, SK-MEL-28, and A375) and colon cancer cell line HCT15 (positive control). (B,C) The expression levels of TOPK dependent on the tumor type (primary melanoma patients, metastatic melanoma patient, and normal person) or gender from The Cancer Genome Atlas-Skin Cutaneous Melanoma (TCGA-SKCM) cohort database. TOPK transcript expression was analyzed according to tissue type according to transcript per million (TPM) units. (D) Effect of 3-DSC on TOPK phosphorylation. Kinase assay was performed with active TOPK (100 ng), 3-DSC (2.5, 5, 10, or 20 μM), HI-TOPK-032 (5 μM), myelin basic protein (MBP) (400 ng), and ATP (40 μM). MBP is the substrate of TOPK kinase; p-Thr/Ser levels are an indicator of TOPK activity. The band intensity of p-Thr/Ser is significantly reduced when the assay was performed with inclusion of 2.5 μM 3-DSC. (E) Band quantification results of (D). (F) Kinetic analysis of 3-DSC whether competing ATP with TOPK substrate. Km and Vm values of TOPK enzyme incubated with or without 3-DSC. (G) Binding affinity of 3-DSC to TOPK was assessed in four skin cancer cells. 3-DSC-Sepharose 4B or Sepharose 4B were incubated with cell lysates. Protein was then eluted from the beads and separated by SDS-PAGE. (H) Diagram illustrating the mutation of residues previously predicted to be important in facilitating the interaction between TOPK and 3-DSC. TOPK was mutated at the T42A, N172A, and T42A residues based upon computer docking analysis. (I) In vitro pull-down assay with TOPK WT, TOPK T42A, TOPK N172A, and TOPK T42A + N172A. Double mutation significantly attenuated binding of 3-DSC with TOPK. *p < 0.05, ***p < 0.001.

FIGURE 2

3-DSC decreases skin epidermal thickening caused by solar ultraviolet (SUV) exposure by targeting TOPK. (A) Representative hematoxylin and eosin (H&E) staining results of mice dorsal skin treated with or without 3-DSC prior to SUV exposure. Each group consisted of 8–10 mice. Tissue thickness was measured using a microscope ruler. Data are shown as mean ± SD values. (B) Protein expressions of p-TOPK, p-ERK, p-RSK, and p-c-Jun were detected by immunohistochemical (IHC) analysis. (C) Quantification of the IHC results in panel (B). Data are shown as mean ± SD values. ***p < 0.001.

FIGURE 3

3-DSC inhibits anchorage-dependent and independent growth in skin cancer cell lines. (A) The inhibition effects of different concentrations of 3-DSC in cell proliferation of A431, SK-MEL-2, SK-MEL-28, and A375 cells. Skin cancer cells were incubated with 3-DSC for 24, 48, 72, and 96 h and growth was detected by MTT assay. Data shown as mean ± SD values. (B) Representative images of anchorage-independent soft agar growth assay using skin cancer cell lines incubated with various concentrations of 3-DSC. (C) Representative images showing the effect of 3-DSC on anchorage-independent growth of EGF-induced transformation of normal skin cells. (D) Quantification of colony numbers shown in panel (C). Data are shown as mean ± SD values. (E) SK-MEL-2 cell anchorage-independent colony growth after knocking down TOPK and treating with various concentrations of 3-DSC. Colony number is quantified in the right side of the panel. (F) A375 cell anchorage-independent colony growth after knocking down TOPK and treating with various concentrations of 3-DSC. Colony number is quantified in the right side of the panel. ***p < 0.001.

FIGURE 4

3-DSC induces arrest at G2/M phase and cell apoptosis in skin cancer cells. (A) Effect of 3-DSC to skin cancer cells’ cell cycle. (B) G2/M phase marker cyclin B1 protein expression level in skin cancer cell lines after treatment with 3-DSC at various concentrations. (C) 3-DSC induces apoptosis in skin cancer cell lines. Skin cancer cells were incubated with various concentrations of 3-DSC for 48 h and analyzed for apoptosis markers indicating pre/late apoptosis by flow cytometry. Data are shown as mean ± SD values. (D) Cell apoptosis marker cleaved PARP/cleaved caspase 3/cleaved caspase 7/PARP/caspase 3/caspase 7 expression after 3-DSC treatment. 3-DSC treatment increased cleaved PARP/cleaved caspase 3/cleaved caspase 7 protein expression in a dose-dependent manner. ***p < 0.001.

FIGURE 5

3-DSC inhibited TOPK signaling pathway in skin cancer cell lines. (A) p-TOPK, TOPK, and downstream effector protein expression after 3-DSC treatment. SK-MEL-2 and A375 cells were incubated with a culture medium with FBS and 3-DSC (5, 10, or 20 μM) or without 3-DSC for 2 h, and cell lysates were harvested before analysis by western blot. (B) TOPK was overexpressed in HEK-293T cells using pcDNA4.1-HisC (control) and pcDNA4.1-HisC-TOPK before cells were treated with or without 3-DSC at various concentrations. (C) Effect of 3-DSC on anchorage-independent growth in HEK-293T cells expressing pcDNA4.1-HisC and pcDNA4.1-HisC-TOPK. TOPK over-expression increased colony number and size. (D) Quantification of colony number in panel (C). Data are shown as mean ± SD values. ***p < 0.001.

FIGURE 6

3-DSC inhibited skin cancer cell-derived xenograft (CDX) growth by attenuating TOPK expression. (A) Bodyweight of three groups of CDX mice over the duration of 43 days. Body weight was measured twice a week. (B) Tumor volume of three groups CDX mice over the duration of the experiment. Tumor size was measured twice a week. (C) Photograph showing tumors excised from control and 3-DSC-treated groups after euthanasia. (D) Quantification of tumor weight in panel (C). Data are shown as mean ± SD values. (E) IHC results of CDX tumor tissues (Ki-67/p-TOPK/p-ERK/p-RSK/p-c-Jun) derived from control and 3-DSC-treated groups. (F) Quantification of IHC results shown in panel (E). Data are shown as mean ± SD values. ***p < 0.001.