Researchers report cancer as the worldwide leading cause of death. An estimated 609,360 people in the US will die from cancer in 2022, corresponding to almost 1,700 deaths/d. The greatest number of deaths are from cancers of the lung, prostate, and colorectum in men and of the lung, breast, and colorectum in women. More than 350 people will die each day from lung cancer, which is more than the corresponding numbers for breast, prostate, and pancreatic cancers combined [1]. Population aging and changing lifestyle are the expected causes for rapidly increasing cancer cases and deaths [2]. Conventional treatments for cancer include surgery, chemotherapy, radiotherapy, immunotherapy, and targeted therapy [3]. In terms of control via conventional cancer treatments, radiotherapy can achieve histologically local control in various cancer types by producing ROS and inducing DNA damage. Despite using advanced radiotherapy, the radiation from radiotherapy often affects the normal tissues [4, 5]. Simultaneously, resistance to radiation often develops in patients receiving radiotherapy, which is one of the main obstacles to cancer treatment.
According to studies, inhibition of
The mitogen-activated protein kinase (MAPK) signaling pathway activity changes the expression of proteins that regulate the adhesion, movement, differentiation, and proliferation of tumor cells; the pathway becomes a significant molecular target for ionizing radiation therapy [7, 8].
MEK is the central downstream component of
KZ-001 is a small-molecular MEK inhibitor developed by us, which is still in the early stage [14]. Preclinical studies have shown that the efficacy of KZ-001 is 30-fold greater than that of AZD6244 in
A549 NSCLC cells (Chinese Academy of Medical Sciences and Peking Union Medical College) were cultured in F12 (Gibco, C11765500BT) medium supplemented with 10% fetal bovine serum (FBS) (Yeasen, 40130ES76), and H460/H441 was maintained in a 1640 (Gibco) medium supplemented with 10% FBS (Yeasen, 40130ES76). Both culture media contained 1% penicillin and streptomycin (Solarbio, P1400). Cellular proliferation assay analysis was conducted using 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) (Solarbio, M1080). In brief, the cells were seeded in 96-well plates (Corning, 3599) at an appropriate density in a 90 μL cell culture medium until attached. Then, KZ-001 (Kechow; the characterization and purity of the KZ-001 are indicated in
RIPA buffer (R0020), protein phosphatase inhibitor (all-in-one, 100×) (P1260), and BCA protein assay kit (PC0020) were purchased from Solarbio. In addition, NuPage 10% Bis-Tris Gel (NP0301BOX) was purchased from Invitrogen. PVDF membrane (Immobilon-P) and eECL western blot kit (CW0049) were from Millipore and CWBIO, respectively. Further, anti-p-ERK1/2 antibody (4370) and anti-ERK1/2 (4695) antibody were purchased from Cell Signaling Technology. Anti-Bcl-xl antibody (ab32370) and anti-Bax antibody (ab32503) were purchased from Abcam. Finally, anti-GAPDH antibody (HRP-60004) was purchased from ProteinTech.
The cells were seeded at an appropriate density (80%–90% confluency). After being subject to treatment with 6 Gy irradiation, KZ-001 at 100 nM, or 6 Gy in combination with KZ-001 at 100 nM, respectively, the cell lysates were extracted and collected using radioimmunoprecipitation assay (RIPA) buffer containing protein phosphatase inhibitor (all-in-one, 100×). As a control, the cells were treated with a 0.1% DMSO medium.
Equal quantities of total protein from cell were resolved on NuPage 10% Bis-Tris Gel and then transferred to the PVDF membrane. The proteins of interest were blotted with the indicated antibodies (anti-p-ERK1/2 ab; anti-ERK1/2 ab; anti-Bcl-xl ab; and anti-Bax ab). As a final step, the chemiluminescence was generated with an eECL western blot kit and detected with the use of a Bio-Rad ChemiDoc XRS+ System.
The A549 and H460 (Chinese Academy of Medical Sciences and Peking Union Medical College) cells were trypsinized to generate a single-cell suspension and were then seeded in 6-well plates for 14 h to allow attachment before treatment. Each treatment condition had 3 replicates. After treating by vehicle (dimethylsulfoxide) or KZ-001 (100 nM) for 1–2 h, the cells were treated with 0 Gy, 1 Gy, 2 Gy, 4 Gy, or 6 Gy of radiation. The drugs were washed out after 24 h; next, the cells were maintained for 2 weeks. The colonies thus obtained were fixed and stained with the use of 0.1% crystal violet (Solarbio, C8470) in 20% methanol. All the colonies consisting of at least 50 cells were counted. Finally, the data were analyzed using GraphPad Prism 8.0 (GraphPad Software, Inc.).
A549 and H460 cells at a density of 2 × 105/well were inoculated into the wells of 6-well plates and cultured overnight. Next, 100 nM KZ-001 or vehicle was added to the indicated wells. Each treatment was triplicated. Subsequently, 100 nM KZ-001 was added to each well, and the vehicle control group was treated with a 0.5% DMSO culture medium. Then, cells were exposed to IR at a dose of 6 Gy. After 24 h, 48 h, and 72 h of treatment, the cells were digested and stained with fluorescein isothiocyanate (FITC), annexin V, and propediene iodide (PI) as per the instructions of apoptosis detection kit (YEASEN, 40301ES50) and analyzed with the use of flow cytometry (BD Biosciences, FACSCelesta).
A549 cells at a density of 1 × 105 cells/mL were seeded into wells of the 6-well plates, cultured overnight, and then pretreated with 100 nM KZ-001 for 1 h. The vehicle control group was treated with a 0.5% DMSO culture medium. Then, these pretreated cells were exposed to IR at a 6 Gy dose. After 48 h incubation, the cells were digested and washed with PBS, fixed in 70% ice-cold ethanol at 4 °C overnight, and then processed in compliance with the instructions accompanying the cell cycle analysis kit (Yeasen, 40301). Ultimately, the fluorescence intensity of each sample was measured using flow cytometry (BD Bioscience, FACSCelesta) and analyzed with ModFit LT software.
A549 cells at a density of 5 × 105/well were seeded into wells of 12-well plates with coverslips (Solarbio, YA0351) and cultured overnight. The next day, the cells were pretreated with 100 nM KZ-001 or 0.5% DMSO for 1 h, and then irradiated with a 6 Gy dose. After 48 h, the cells were fixed with a 10% neutral buffered formalin (Solarbio, G2161) at 37 °C for 30 min and permeabilized with Triton X-100 (Solarbio, P1080) for 30 min at room temperature. After being blocked with PBS containing 1% bovine serum albumin (Solarbio, A8010) at room temperature for 60 min, the samples were incubated with primary rabbit anti-gamma H2AX (phospho S139) antibody (Abcam, ab81299; 1:250) at 4 °C overnight followed by the anti-rabbit-PE secondary antibody (Proteintech, SA00008-2; 1:50) at room temperature for 1.5 h in the dark.
Statistical analyses were performed with the use of GraphPad Prism 8.0 (GraphPad Software, Inc.), using
We used different concentrations (0.5–1,000 nM) of KZ-001 to treat 3 different NSCLC cells for 24 h, 48 h, and 72 h. The results illustrated that KZ-001 had a strong inhibitory effect on the A549 and H460 proliferation for 72 h (
Next, we used the MTT method for a preliminary exploration of the radiosensitivity of the cells.
Two NSCLC cell lines, A549 and H460, were pretreated with 100 nM KZ-001 for 1 h before irradiation. After 2 weeks, the number of clones was counted. Results showed that KZ-001 caused the radiosensitivity under 1 Gy, 4 Gy, and 6 Gy irradiation doses. However, KZ-001 did not aggravate the radiation impairment of H460 under any irradiation doses (
We used annexin V and PI double staining method after exposure to 6 Gy irradiation dose with or without KZ-001 treatment to detect A549 and H460 apoptosis.
To further explore the mechanism of apoptosis at the molecular level, we detected the anti-apoptotic marker Bcl-XL and the apoptotic marker Bax in the A549 cells (
Generally, one common property of
Since it was observed that the combination of KZ-001 with IR promoted the radiosensitivity on A549, we further investigated whether KZ-001 can affect the cell cycle arrest. In the experiment, A549 was pretreated with 100 nM KZ-001 for 1 h and then exposed to 6 Gy irradiation dose. After 48 h, the cells were collected, stained with PI, and examined for changes in the cell cycle distribution using a flow cytometer. The outcome showed that compared to control, the irradiation alone increased the proportion of cells in the G2 phase from 9.14% to 21.24%, and the combination of IR and KZ-001 could further increase the proportion to 32.22% (
IR could cause DNA damages to the tumor cells, and DNA double-strand breaks (DSBs) are the most crucial form of this damage. DSBs subsequently trigger the DNA repair mechanism. Activated γH2AX is a significant biomarker for the DNA repair process; hence, γH2AX (the number of phosphorylated γH2AX nuclear aggregates) activation extent can measure the degree of DNA damage caused by IR. Detection results of the phosphorylated γH2AX in cells showed that compared to the irradiation group, the KZ-001 could significantly increase the positive cell ratio to 33.4% (
Lung cancer is the leading cause of cancer-related death worldwide, and NSCLC accounts for 85% of all lung cancers. NSCLC has a poor prognosis. In addition, NSCLC is resistant to radiotherapy, partly due to various genetic changes in NSCLC, including mutation, amplification, deletion, and fusion, which aggravates the signal pathway and physiological activity abnormalities [15, 16]. It is worth noting that 20%–30% of NSCLC has a
We evaluated the radiosensitivity effect of KZ-001 in 2
The findings of our study demonstrate that the 2 types of NSCLCs showed different radiosensitivity, and we next investigated the association between KZ-001 and IR-mediated apoptotic cell death in these NSCLCs. Interestingly, the KZ-001 with IR-induced apoptotic ratio in A549 and H460 was significantly increased in comparison with that induced by IR monotherapy alone. The H460 cell line apoptotic ratio is statistically significant, while it lacks biological significance compared to the A549 cell line apoptotic ratio. KZ-001-mediated downregulation of Bcl-XL could also support this phenomenon in both A549 and H460. Although we did not observe the upregulation of Bax within 24 h after the KZ-001 treatment in A549, the upregulation of Bax was inevitable with the prolongation of KZ-001 action.
DNA damage via ionizing radiation is one of the key approaches to kill cancer cells; in fact, DNA DSB is the most crucial type of DNA damage and the decisive factor for cell radiosensitivity. Histone γH2AX phosphorylation is a DNA damage marker, especially when DNA DSBs are involved [21]. We used fluorescence immunoassay to evaluate the KZ-001 effect on the level of DNA damage marker γH2AX. As expected, in A549 cell lines, the combination of KZ-001 and radiotherapy significantly increased the level of intracellular γH2AX phosphorylation compared to radiation alone.
DNA damage response can cause cell cycle arrest by activating cell cycle checkpoints, promoting DNA repair, and changing transcription, and ultimately maintaining cell survival. Studies have shown that CHK1 is phosphorylated and activated when DNA undergoes damage and blocks the cell cycle in the G2/M phase. Our results show that compared to the radiotherapy alone, the KZ-001 combined with radiotherapy had induced more cell cycle arrest in the G2/M phase, indicating that KZ-001 combined with radiotherapy had induced more DNA damage. This was consistent with the immunofluorescence results.
This study shows that radiotherapy combined with MEK inhibitor KZ-001 can significantly block the MAPK pathway, reduce the ERK phosphorylation level, significantly increase DNA DSBs, increase cell cycle G2/M phase arrest, induce A549 apoptosis, and finally play a role of radiosensitizer. Therefore, the MEK inhibitor KZ-001 is a potent radiosensitizer for future clinical applications.