KMU-191 Induces Apoptosis in Human Clear Cell Renal Cell Carcinoma Caki Cells Through Modulation of Bcl-xL, Mcl-1 (L), c-FLIP (L), and p53 Proteins

The anti-proliferative effects of a newly developed N3-acyl-N5-aryl-3,5-diaminoindazole analog, KMU-191, have been previously evaluated in various cancer cells. However, the detailed anti-cancer molecular mechanisms of KMU-191 remain unknown. In this study, we investigated anti-cancer mechanisms by which KMU-191 regulates apoptosis-related genes in human clear cell renal cell carcinoma Caki cells. KMU-191 induced poly ADP-ribose polymerase cleavage and caspase-dependent apoptosis. In addition, KMU-191 induced down-regulation of the long form of cellular FADD-like IL-1β-converting enzyme inhibitory protein (c-FLIP (L)) at the transcriptional level as well as that of long form of myeloid cell leukemia (Mcl-1 (L)) and B-cell lymphoma-extra large at the post-transcriptional level. Furthermore, KMU-191-induced apoptosis was closely associated with the Mcl-1 (L) down-regulation, but also partially associated with c-FLIP (L) down-regulation. In contrast, KMU-191 up-regulated p53, which is closely related to KMU-191-induced apoptosis. Although KMU-191 showed cytotoxicity of normal cells, it unusually did not induce cardiotoxicity. Taken together, these results suggest that a multi-target small molecule, N3-acyl-N5-aryl-3,5-diaminoindazole analog, KMU-191 is a potential anti-cancer agent that does not induce cardiotoxicity.


Introduction
The discovery of aminoindazole-based anticancer drugs that target several kinases and elucidation of their anti-cancer mechanisms are widely pursued to establish a scientific basis for their anti-cancer property [1][2][3][4]. Previous study has revealed that a novel compound, N3-acyl-N5aryl--3,5-diaminoindazole analog (KMU-191) has anti-proliferative effects in various cancer cell lines [5]. However, the anti-cancer mechanisms of  have not been fully investigated. In this study, we demonstrated that anti-cancer mechanisms of KMU-191 are associated with apoptosis in human clear cell renal cell carcinoma cells.
Apoptosis, an important programmed cell death process, is involved in various biological processes, including homeostasis through normal cell turnover,

Ivyspring
International Publisher adequate embryonic development, immune system function, and elimination of unwanted or damaged cells [6][7][8][9]. Apoptosis occurs through two major pathways: the extrinsic pathway, in which components such as FLIP and caspase-8 are involved, and the intrinsic pathway in which the bcl-2 family is involved, such as B cell lymphoma-2 (Bcl-2), B-cell lymphoma-extra large (Bcl-xL), and myeloid cell leukemia 1 (Mcl-1) [10,11]. In addition, the tumor suppressor gene p53 plays an important role in apoptosis in response to DNA damage [12].
Unexpected cardiotoxicity is a critical cause of the termination of clinical trials and drug withdrawal from the market [13]. Torsade de pointes (TdP), a specific form of polymorphic ventricular tachycardia, prolongs the QT interval by inhibiting ion channels associated with the action potential duration in the myocardial cell membrane and inducing cardiotoxicity associated with the side effects of chemicals [14]. Predicting TdP incidence is important for assessing cardiotoxicity and safety in the development of new drugs. Therefore, using electrocardiography (ECG) and left ventricle pressure (LVP), it is crucial to ensure the electrophysiological safety of drugs based on a reliable assessment of their proarrhythmic cardiotoxicity potential [15,16].
This study aimed to investigate the molecular mechanism underlying the anti-cancer effect of KMU-191 and its electrophysiological safety for the first time. The results revealed that KMU-191 induces apoptosis in human clear cell renal cell carcinoma Caki cells through the modulation of Mcl-1 (L), c-FLIP (L), and p53, and had no effect on cardiotoxicity.

Sulforhodamine B (SRB) assay
After the indicated incubation time, adherent cell cultures were fixed in situ by adding 50 µL of 50% (w/v) cold trichloroacetic acid (Sigma Chemical Co., St. Louis, MO, USA) and incubated for 60 min at 4 °C. The supernatant was discarded and the plates were washed five times with deionizedwater and dried. Fifty microliters of SRB (Sigma Chemical Co., St. Louis, MO, USA) solution (0.4%w/v) in 1% acetic acid (PanReac AppliChem, Barcelona, Spain) were added to each well and incubated for 30 min at room temperature. Plates containing SRB solution were washed five times with 1% acetic acid. Then, plates were air dried and 100 µL/well of 10 mM Tris base (pH 10.5) (Sigma Chemical Co., St. Louis, MO, USA) were added and the absorbance of each well was read on an enzyme-linked immunosorbent assay reader at 510 nm. Finally, cytotoxicity was measured as the percentage of absorbance compared to that of control cells.

Western blotting analysis
Cellular lysates were obtained by suspending 0.5 × 10 6 cells in 80 µL of RIPA buffer (20 mM HEPES and 0.5% Triton X-100, pH 7.6). The cells were disrupted by vortexing and extracted at 4 °C for 30 min. Protein concentrations were evaluated using the BCA assay kit (Thermo Fisher Scientific, Waltham, MA, USA). Proteins were electrotransferred onto Immobilon-P membranes (Millipore Corp., Bedford, MA, USA). Specific proteins were detected using an ECL Western blotting kit (EMD Millipore, Darmstadt, Germany) according to the manufacturer's instructions. The signal strength was analyzed using a Chemi Image System Fusion FX (Vilber Lourmat, Collégien, France).

Flow cytometric analysis
Approximately 0.3 × 10 6 cells were suspended in 100 µL Phosphate-buffered saline (PBS), and 200 µL of 95% ethanol was added while vortexing. The cells were incubated at 4 °C for 1 h, washed with PBS, and resuspended in 250 µL of 1.12% sodium citrate buffer (pH 8.4) along with 12.5 µg RNase. Incubation was continued at 37 °C for 30 min. Cellular DNA was then stained by adding 250 µL propidium iodide (50 µg/mL) for 30 min at room temperature. Stained cells were analyzed using a FACScan flow cytometer (Becton Dickinson and Co., Franklin Lakes, NY, USA) to determine the relative DNA content based on red fluorescence.

RNA isolation and quantitative real-time PCR (qPCR)
Total cellular RNA was extracted from the tissues using TRIzol reagent (Molecular Research Center, Inc., Cincinnati, OH, USA). RNA was quantified using Nanodrop 1000 (Thermo Scientific, Wilmington, DE, USA). Each cDNA was synthesized from 2 µg of total RNA using M-MLV reverse transcriptase (Promega, Madison, WI, USA) according to the manufacturer's protocol. Table 1 lists the specific primer pairs and SYBR GREEN Premix used (TOYOBO, Japan). qPCR was performed on the LightCycler ® 480 real-time PCR system (Roche Diagnostics, Mannheim, Germany). β-actin was used as a housekeeping gene for normalization, and a no-template sample was used as a negative control. Then, the qPCR data were analyzed by the 2 −ΔΔct method [20].

Small interfering RNA (siRNA) Transfection
p53 siRNA duplexes were obtained from Cell Signaling Technology (Danvers, MA, USA). The control siRNA duplexes used in this study were purchased from Invitrogen (Carlsbad, CA, USA) and had the following sequence: green fluorescent protein, AAGACCCGCGCCGAGGUGAAG. The cells were transfected with siRNA oligonucleotides using Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA, USA) per the manufacturer's instructions.

Langendorff heart preparation
Sprague Dawley rats of male (200 -250 g) were injected intravenously with 1000 IU/kg heparin and then anesthetized by intravenous injection of 30 mg/kg sodium pentobarbital. After they were sedated and had lost pedal reflex activity, the hearts were rapidly excised and perfused via the aorta on a Langendorff apparatus (EMKA Technologies, Paris, France) using modified Krebs-Henseleit buffer saturated with carbogen (95% O2 and 5% CO 2 ) containing 112 mM NaCl, 5 mM KCl, 11.5 mM glucose, 25 mM NaHCO 3 , 1.2 mM MgSO 4 , 1.2 mM KH 2 PO 4 , 2 mM pyruvic acid, and 1.25 mM CaCl 2 at a constant perfusion pressure (60 -80 mmHg). All the experiments were conducted after stabilization for 10 min.

LVP and ECG recordings
To measure LVP, a water-filled latex balloon attached to a metal cannula was placed in the left ventricle through the pulmonary vein and connected to a pressure transducer (EMKA Technologies, Paris, France). ECGs were recorded using two surface electrodes (EMKA Technologies, Paris, France) held with a spring against the epicardium. One electrode was placed on the right ventricle near the atrium-ventricle ring and the second on the left ventricle in a position. All hemodynamic parameters were recorded and evaluated using iox2 software (EMKA Technologies, Paris, France) for 10 -15 min before and after reperfusing KMU-191 with buffer. The QTc in milliseconds was calculated as follows: Bazett: QTcB = QT/RR 1/2 ; Fridericia: QTcF = QT/RR 1/3 . KMU-191 was perfused serially from low concentration (1 μM) to high concentration (10 μM). The procedures used in this study were reviewed and approved by the Institutional Animal Care and Use Committee of the Korea Institute of Toxicology (RS21006).

Statistical analysis
All data were statistically analyzed by one-way analysis of variance followed by post hoc comparisons (Student-Newman-Keuls test) using SPSS (ver. 27.0) software (SPSS, Inc., Chicago, IL, USA). Statistical significance was set at P-value < 0.05.

Anti-cancer effect of KMU-191 in various human cancer cells
Previously, we found that KMU-191 has an anti-proliferative effect on various human cancer cells [5]. This study was designed to investigate the anti-proliferative effect of KMU-191 on various human cancer cell lines using the SRB assay. As shown in Figure 1B Figure 4B). Next, to investigate the functional role of p53 in KMU-191-mediated apoptosis in cancer cells, we used Caki cells transfected with siRNA targeting p53 mRNA and HCT116 cells (p53 wild-type and p53 deficient). Immunoblot analysis demonstrated that transfection with p53 siRNA suppressed p53 expression in Caki cells compared to that in cells transfected with control GFP siRNA ( Figure 4C). Remarkably, KMU-191mediated apoptosis and PARP cleavage were inhibited in Caki cells transfected with p53 siRNA compared to those in control GFP siRNA-transfected Caki cells ( Figure 4C). Moreover, apoptosis and PARP cleavage were inhibited in KMU-191-treated p53 deficient HCT116 cells, but not in p53 wild-type HCT116 cells ( Figure 4D).  To explore MAPK signaling, which plays an important role in cell proliferation, survival, and differentiation [21,22], during KMU-191-induced apoptosis, we used specific MAPK inhibitors. However, none of the specific MAPK inhibitors (PD, MEK inhibitor; SP, JNK inhibitor; SB, p38 MAPK inhibitor) attenuated KMU-191-induced apoptosis in Caki cells ( Figure 4E). Reactive oxygen species (ROS), byproducts of normal oxygen metabolism, play an important role in apoptosis under both physiologic and pathologic processes [23]. Therefore, we investigated the role of ROS in the apoptosis of Caki cells induced by . As shown in Figure 4F, KMU-191-induced apoptosis was not suppressed by pretreatment with NAC and GEE. These results demonstrated that KMU-191-induced apoptosis is not associated with either MAPK signaling pathways or ROS.

Modulation of Mcl-1 (L), Bcl-2, and c-FLIP (L) expression levels in KMU-191-treated Caki cells
To evaluate whether KMU-191 have cytotoxic effect in normal cells, we used TCMK-1 and MC. KMU-191 induced apoptosis of TCMA-1 and MC cells in a dose-dependent manner ( Figure 4G and 4H). In addition, KMU-191 induced PARP cleavage, up-regulation of p53 and p21 in TCMK-1 cells ( Figure  4G). These results suggested that the cytotoxicity of KMU-191 is induced by similar mechanisms in cancer cells and normal cells.

The effect of KMU-191 on cardiotoxicity assessment
Many anticancer drugs cause cardiotoxicity as a side effect [24,25]. We assessed cardiotoxicity in a dose-dependent manner (1 -10 µM) to explore whether KMU-191 causes cardiotoxicity. To assess cardiotoxicity, we used the Langendorff heart system for LVP and ECG analyses. We analyzed cardiotoxicity factors such as heart rate, developed pressure, maximal rate of contraction and relaxation (dP/dtmax and dP/dt min ), ECG waveform RR Interval, QT Interval, QT correction Bazett (QTcB) Interval, and QT correction Fridericia (QTcF) Interval. As shown in Figure 5A, KMU-191 did not affect any of the hemodynamic parameters in the LVP analysis. In addition, the results of the ECG analysis showed that KMU-191 dose not induce cardiotoxicity in a dose-dependent manner ( Figure 5B). Thus far, these results suggested that KMU-191 has the potential to induce apoptosis of cancer cells without causing cardiotoxicity.
Although targeted anti-cancer therapy has attracted attention as an effective therapeutic strategy [29], there is an unmet need to provide more efficacious cancer treatments due to intratumoral heterogeneity [30]. For this reason, the development of multi-target drugs capable of regulating various targets involved in cancer pathophysiology has received considerable attention [31,32]. Dysregulation of anti-apoptotic Bcl-2 family proteins, such as Bcl-xL and Mcl-1, is associated with the survival of cancer cells and resistance to anti-cancer drugs [33,34]. Moreover, c-FLIP overexpression frequently occurs in various types of human cancers and is associated with inhibition of cell death receptormediated apoptosis [35,36]. As shown in Figure  Nuclear or cytoplasmic accumulation of the tumor suppressor p53 is well known as an important mechanism of DNA damage-induced apoptosis [37]. In this study, KMU-191 induced p53 up-regulation in a dose-and time-dependent manner ( Figure 2C and 2D). Remarkably, p53 siRNA blocked KMU-191induced apoptosis in Caki cells ( Figure 4C). Moreover, KMU-191 induced apoptosis in cancer cells of various status of p53 gene ( Figure 2C, 2E, and 2F). Interestingly, KMU-191 induced G2/M arrest in p53 deficient HCT116 cells ( Figure 2G). These results suggest that p53 up-regulation plays an important role in KMU-191-induced apoptosis and KMU-191 has anti-proliferative effect of cancer cells regardless of p53 status.
Among the apoptosis pathways in cancer cells, ROS generation and the MAPK pathways are well known as important regulators in the establishment of cancer therapeutic strategies [38,39]. Therefore, we tested specific inhibitors to evaluate the involvement of ROS generation and MAPK pathways in KMU-191-mediated apoptosis. However, none of the specific inhibitors reduced KMU-191-induced apoptosis in Caki cells ( Figure 4E and 4F). Cardiovascular toxicity is a major factor limiting the development and clinical application of this class of drugs, such as kinase inhibitors [40]. Cardiovascular toxicity and safety assessments such as LVP and ECG are required to evaluate the possibility of inducing arrhythmia during drug development [41]. Therefore, we determined the cardiovascular toxicities of KMU-191 using ECG and LVP analyses using the Langendorff test. The Langendorff heart preparation method is one of the screening ex vivo models for evaluating the proarrhythmic toxicity of test substance on the heart by excluding external influences from humoral and neural factors [42]. In addition, according to the ICH S7B guideline, which describes a non-clinical testing strategy for assessing the proarrhythmic risk of drugs, the Langendorff heart preparation method is known as a good method to evaluate the proarrhythmic risk of drugs [43]. As shown in Figure 5, KMU-191 had little few potential risks associated with cardiotoxicity, suggesting that KMU-191 has the potential to be developed as an anti-cancer drug.
Our study had some limitations that should be investigated more closely. First, given that KMU-191 is a kinase inhibitor, the mechanism of kinase inhibition of KMU-191 and its relevance to the upand down-regulation of proteins have not been fully explored. Additionally, the sensitivity to KMU-191 among the cells used differed, so it is necessary to compare which genetic backgrounds exhibit differences in sensitivity to . Although KMU-191 had anti-cancer effect in Caki, it had also cytotoxicity in normal cell lines such as TCMK-1 and MC. Finally, we did not validate the anti-cancer efficacy of KMU-191 in vivo. Therefore, further studies are needed to elucidate the deeper mechanisms of anti-cancer effect of KMU-191, and compound optimization studies to reduce cytotoxicity in normal cells are necessary.
Despite the limitations of this study, we found that KMU-191 induced apoptosis in various cancer cells by activating caspase-3, down-regulating Bcl-xL, Mcl-1 (L), and c-FLIP (L), and up-regulating p53. Moreover, we demonstrated the electrophysiological safety of KMU-191 ex vivo. Collectively, these results suggest that KMU-191 is a clinically useful and attractive multi-target agent against cancer.