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, 20 (23)

A Small Compound KJ-28d Enhances the Sensitivity of Non-Small Cell Lung Cancer to Radio- And Chemotherapy

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A Small Compound KJ-28d Enhances the Sensitivity of Non-Small Cell Lung Cancer to Radio- And Chemotherapy

Hwani Ryu et al. Int J Mol Sci.

Abstract

We previously reported on a poly (ADP-ribose) polymerase (PARP) 1/2 inhibitor N-(3-(hydroxycarbamoyl)phenyl)carboxamide (designated KJ-28d), which increased the death of human ovarian cancer BRCA1-deficient SNU-251 cells. In the present study, we further investigated the antitumor activities of KJ-28d in BRCA-proficient non-small cell lung cancer (NSCLC) cells to expand the use of PARP inhibitors. KJ-28d significantly inhibited the growth of NSCLC cells in vitro and in vivo, and induced DNA damage and reactive oxygen species in A549 and H1299 cells. Combined treatment with KJ-28d and ionizing radiation led to increased DNA damage responses in A549 and H1299 cells compared to KJ-28d or ionizing radiation alone, resulting in apoptotic cell death. Moreover, the combination of KJ-28d plus a DNA-damaging therapeutic agent (carboplatin, cisplatin, paclitaxel, or doxorubicin) synergistically inhibited cell proliferation, compared to either drug alone. Taken together, the findings demonstrate the potential of KJ-28d as an effective anti-cancer therapeutic agent for BRCA-deficient and -proficient cancer cells. KJ-28d might have potential as an adjuvant when used in combination with radiotherapy or DNA-damaging agents, pending further investigations.

Keywords: DNA damage; chemotherapy; combination therapy; non-small cell lung cancer; poly (ADP-ribose) polymerase inhibitor; radiotherapy.

Conflict of interest statement

The authors declare that they have no conflicts of interest.

Figures

Figure 1
Figure 1
KJ-28d inhibits tumor growth of A549 and H1299 xenografts in nude mice. (A) The chemical structure of the KJ-28d compound. (B) A549, H1299, H1650, and H460 human non-small cell lung cancer (NSCLC) cells were treated with KJ-28d at the indicated concentrations for 5 days, and cell viabilities were determined by the MTT assay. Data are presented as means ± standard deviation (SD) from at least three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 versus DMSO-treated control. (C,D) A549 cells and H1299 cells were subcutaneously injected into the thigh of the right hind leg of BALB/c nu/nu mice (n = 3 per group, A549; n = 4 per group, H1299). Two weeks after tumor cell injection, KJ-28d (10 mg/kg) or DMSO (control) was intraperitoneally administered once every 2 or 3 days for seven times in total. (C) Longest (L) and shortest (W) tumor axes were measured, and tumor volume (mm3) was calculated as L × W2/2. Data shown represent average tumor volume (* p < 0.05, ** p < 0.01). Results are shown as means ± SD. (D) The body weights of A549 and H1299 xenograft mice were determined once a week during the experiments. Data are shown as means ± SD.
Figure 2
Figure 2
KJ-28d induces DNA damage and generation of reactive oxygen species (ROS). (A) A549 and H1299 cells were treated with 5 μM KJ-28d at indicated time points and immunoblotted for the detection of expression of γ-H2AX. (B,C) A549 and H1299 cells were treated with 5 mM NAC for 1 h, followed by KJ-28d for 24 h after incubation with 2′,7′-dichlorodihydrofluorescein diacetate (CM-H2DCFHDA) for 30 min. Total cellular ROS production was measured using flow cytometry. Data are representative of three independent experiments (B). The bar graph shows the quantitative analysis of flow cytometer data (C). Data are presented as the mean ± SD of three independent experiments. * p < 0.05, ** p < 0.01 versus corresponding values.
Figure 3
Figure 3
KJ-28d potentiates ionizing radiation (IR)-induced DNA damage responses. A549 and H1299 cells were treated with 5 μM KJ-28d 2 h before IR (4 Gy) and incubated for 24 h. The cell lysates were subjected to immunoblotting for detection of γ-H2AX (A), whereas cells were immunostained for γ-H2AX foci (red) and nuclei (DAPI: blue). Images were captured at 400× magnification. Scale bar: 20 μm (B). Quantification of the number of γ-H2AX foci per cell (C). Data represent the mean ± SD of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 versus corresponding cells. (D) H1299 cells were treated with KJ-28d at indicated concentrations for 1 h. (E) A549 cells were treated with 5 μM KJ-28d and IR (4 Gy) and incubated for 1 h. The cell lysates were immunoblotted for the detection of expression of PAR. β-actin was used as a loading control.
Figure 4
Figure 4
KJ-28d enhances the radiosensitivity of A549 and H1299 cells. (A) A549 and H1299 cells were treated with 0.75 μM KJ-28d for 2 h before IR (0, 1, 2, and 4 Gy). Clonogenic survival was measured 10 days after IR. Data are expressed as mean ± SD (n = 3) of the surviving fraction compared to non-irradiated cells. Colonies consisting of more than 50 cells were scored as survival colonies. (B) A549 and H1299 cells were treated with either 5 μM KJ-28d or 5 μM olaparib plus IR (4 Gy) for 48 h. Apoptotic cells were determined using the APC-conjugated annexin V/PI staining. Cell populations were gated into four groups, as described in Section 4. Bar graphs represent the mean percentage of early (annexin V-positive/PI-negative) and late apoptotic cells (annexin V-positive/PI-positive). Data represent the mean ± SD of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 versus corresponding cells. (C) A549 and H1299 cells were treated with 5 μM KJ-28d plus IR (4 Gy), and the cell lysates were subjected to immunoblotting for detection of cleaved caspase-3. β-actin was used as a loading control.
Figure 5
Figure 5
The combination of KJ-28d and DNA damage-inducing chemotherapeutic agents synergistically inhibits growth of A549 and H1299 cells. A549 (A) and H1299 (C) cells were treated with the single or combined administration of KJ-28d and carboplatin, cisplatin, paclitaxel, or doxorubicin at indicated concentrations. Cell viability was determined 5 days after the treatment by MTT assay. Relative viability (normalized to DMSO-treated cells) is shown for each combination at indicated concentrations. Data are from one representative experiment of three independently repeated experiments. (B,D) Summary of tables showing combination index (CI) scores of KJ-28d and each chemotherapeutic drug combined at indicated concentrations in A549 (B) and H1299 cells (D). CI scores were calculated using the CompuSyn software and categorized as synergistic (CI < 0.9, green), additive (1.1 > CI ≥ 0.9, blue), or antagonistic (CI ≥ 1.1, gray). Each CI score was one representative datum from treatment with the indicated concentrations of single- and paired compounds from more than three independent experiments.

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