KAN0438757: a novel PFKFB3 inhibitor that induces programmed cell death and suppresses cell migration in non-small cell lung carcinoma cells

doi:10.21203/rs.3.rs-3067846/v1

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KAN0438757: a novel PFKFB3 inhibitor that induces programmed cell death and suppresses cell migration in non-small cell lung carcinoma cells

https://doi.org/10.21203/rs.3.rs-3067846/v1

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Background

PFKFB3 is glycolytic activators that is overexpressed in human lung cancer and plays a crucial role in multiple cellular functions including programmed cell death. Despite the many small molecules described as PFKFB3 inhibitors, some of them have shown disappointing results in vitro and in vivo. On the other hand KAN0438757, selective and potent, small molecule inhibitor has been developed. However, the effects of KAN0438757, in non-small cell lung carcinoma cells (NSCLCs) remain unknown. Herein, we sought to decipher the effect of KAN0438757 on proliferation, migration, DNA damage, and programmed cell death on NSCLCs.

Methods and Results

The effects of KAN0438757 on cell viability, proliferation, DNA damage, migration, apoptosis, and autophagy in human NSCLC cells was tested by WST-1, real-time cell analysis (RCTA), colony formation, comet assay, wound-healing migration test, and MMP/JC-1 and AO/ER dual staining assays as well as western blot analysis. Results revealed that KAN0438757 significantly suppressed the viability and proliferation of A549 and H1299 cells and inhibited colony formation and migration of A549 cells. More importantly, KAN0438757 caused DNA damage and triggered apoptosis and this was accompanied by the up-regulation of cleaved PARP in A549 cells. Furthermore, treatment with KAN0438757 resulted in increased LC3 II and Beclin1, which indicated that KAN0438757 stimulated autophagy.

Conclusions

Overall, targeting PFKFB3 with KAN0438757 may be a promising effective treatment approach, requiring further in vitro and in vivo evaluation of KAN0438757 as a therapy in NSCLCs.

PFKFB3

KAN0438757

Apoptosis

Autophagy

Lung Cancer

Transformed cells more than non-transformed cells exert heavy demands on glycolysis to support their increased energy consumption owing to their rapid rates of cell division. The glycolysis pathway is the basic enzymatic process in cell metabolism. However, under limited oxygen, normal cells respond physiologically by increasing glycolysis. The dysregulation of glucose metabolism is one of the hallmarks of cancer cells. High glucose consumption by tumors was first reported by Warburg in the 1920s and later suggested that cancer cells exhibit a respiratory disorder [1, 2]. Cancer cells choose the aerobic glycolysis (known as “Warburg” effect), which is an efficient way of rapidly synthesizing nucleotides, amino acids and lipids required to form biomass [3]. 6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatases (PFKFBs) are enzyme family, best known for its role in the glycolysis. PFKFBs enzyme family consists of four isoforms are encoded by separate genes: PFKFB1, PFKFB2, PFKFB3, and PFKFB4 [4]. Among these members, the PFKFB3 enzyme exhibit kinase activity higher than phosphatase activity, has the highest Kinase/Biphosphatase activity ratio (740-fold) expressed by PFKFBs [5]. The essential activity of PFKFB3 is to catalyze the synthesis of F2,6BP, which subsequently activates phosphofructokinase-1 (PFK-1) that enables upregulating of the glycolytic flux [6]. Previous findings indicated that PFKFB3 can be regulated by several tumor-related genes, including phosphatase and tensin homolog (PTEN) [7], mitogen activated protein kinase (MAPK) [8], phosphoinositide 3-kinases (PI3K/Akt) [9], and hypoxia-inducible factor 1-alpha (HIF-1a) [10], in cancer cell lines. However, in spite of this abundance of potential targets in glycolytic flux, PFKFB3, has received considerable attention. In recent years, potent and selective PFKFB3 inhibitors have been identified and investigated as potential anticancer agents. 3PO (3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one), a chalcone group, is the first small-molecule inhibitor of PFKFB3 in this class to be evaluated in vitro and human-derived tumor studies. 3PO has demonstrated impressive single-agent activity via prohibit tumorigenic growth of breast , leukemia, and lung adenocarcinoma cells [11]. Moreover, it exhibits synergistic activity when administered in combination with a multi-kinase inhibitor Sunitinib L-malate in preclinical studies in Human endothelial cells (HUVECs) [12]. On the other hand, PFK15, also a potent and selective PFKFB3 inhibitor, displayed a more (aprx.100-fold) potent effect than 3PO in preclinical studies [13]. PFK15 inhibit cell migration and invasion in HNSCC cell culture and block the metastasis to lung in the xenograft mice models [14]. PFK158, an PFK15-based synthetic inhibitor assessed through active phase I clinical trials, exhibits a high affinity to the ATP binding site and elicits a significant antitumor effect on lung, glioblastoma, melanoma, pancreatic, and colon cancer. In more recent study, Gustafsson et al. revealed that RNAi or pharmacological inhibition of PFKFB3 is involved in DNA damage response and deoxynucleotide incorporation upon DNA repair. In addition, the results showed that PFKFB3 inhibition plays a critical role in homologous recombination and defective nucleotide incorporation. KAN0438757 is a newly developed small molecule inhibitor of PFKFB3 that impairs DNA repair by disrupting deoxynucleotide incorporation and inhibits survival of transformed cells [15]. Previous studies have found that PFKFB3 expression is increased significantly in some cancers [16] including lung cancer; however, whether KAN0438757, a novel and specific small molecule inhibitor of PFKFB3, has anti-neoplastic activity on lung cancer is unknown. Since lung cancer is very resistant to chemotherapy, and PFKFB3 has been shown to crucial effects in its survival, the objective of the study was to reveal the potential therapeutic role of KAN0438757 by targeting PFKFB3.

In this study, we found that the pharmacological inhibition of PFKFB3 by KAN0438757 effectively suppressed the proliferation and colony formation ability of lung cancer cells. It also arrested the migration and induced DNA damage and apoptosis in vitro. Furthermore, KAN0438757 distrupted Mitochondrial Membrane Potential (MMP) and induced Autophagic biomarkers; LC3 and Beclin1. Overall, KAN0438757 treatment may be a novel approach to suppressing Lung cancer.

Main reagents, Antibodies, and KAN0438757

Protease-phosphatase inhibitor cocktail, trypan blue, Tris HCl, trypsin-EDTA, dimethyl sulfoxide (DMSO), TEMED, Sodium azide (NaN3), luminol, Skim milk powder, P-coumaric acid, bovine serum albumin (BSA) and others was obtained from Sigma Aldrich company. Dulbecco’s modified Eagle’s medium (DMEM) fetal bovine serum (FBS), penicillin/streptomycin, and phosphate buffered saline (PBS) were purchased from Gibco. WST-1 assay was obtained were obtained from Boster (USA). GAPDH, p62, Beclin 1, LC3, PARP antibodies used in Western blot studies were obtained from SantaCruz Biotechnology (USA), and Anti-mouse antibody was obtained from Invitrogen (USA).

PFKFB3 inhibitor KAN0438757 (Cat No: S0400) was purchased from Selleck Chemicals GmbH (Houston, TX 77014 USA). It was dissolved in sterile dimethyl sulfoxide (DMSO, sterile-filtered, suitable for cell culture) at a final concentration of 1 mM, then stored in aliquots at −80°C and working concentrations were diluted in culture medium.

Cell Lines and Culture Conditions

All cell lines used (A549, H1299, and COS7) cell lines were provided by ATCC (American Type Culture Collection, Manassas, USA). A549 and COS7 were maintained in DMEM (Dulbecco’s modified Eagle’s medium containing with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (Gibco, Grand Island, NY, USA). H1299 cells were cultured in RPMI (Roswell Park Memorial Institute) 1640 medium added with 10% FBS and 1% penicillin-streptomycin. Cell lines used in this study were grown by culturing at 37 °C, 95% humidity and 5-6% CO2.

Cell Viability

The viability of A549, H1299, and COS7 cells was assessed with the WST-1 assay kit (Boster, USA). Briefly the cells were treated with KAN0438757 at concentrations of 1, 5, 10, 25, 50, and 100 µM) and time point (24, 48, 72h). Then, 10μl WST-1 solution was added into the wells. The plates were kept out of light and incubated in a 37°C CO₂ (5%) incubator for 3h. Afterthat, it was measured spectrophotometrically at 420-480 nm using an ELISA microplate reader (Spectra Max 384 Plus).

Real-Time Cell Analysis (RCTA)

A549 and H1299 cells (1.5×10⁴ cells/well ) and A549 cells (1×10⁴ cells/well) were plated into the E-Plate16 and then incubated in xCELLigence RTCA instrument (ACEA Bioscience, CA, USA) at 37°C and 5% CO₂. E-16 plates have microelectrodes to measure the cellular impedance which was recorded every 15 min for 96 h by xCELLigence RTCA. After attaching them to the plate, the cells were given different doses of KAN0438757 and then incubated for 48 hours. Recorded data were analyzed by RTCA Software (v1.2, ACEA Biosciences Inc.).

Clonogenic Survival Assay

Clonogenic survival assay was performed as described earlier [24]. Briefly, in this experiment, the cells were seeded on six-well plates at 800 cells/well, and cultured overnight, after which they were incubated with indicated doses of KAN0438757 for 48 h. After 12 days of treatments, cells were fixed with methanol-acetic acid for 12 minutes and stained with 0.5% crystal violet for 15 minutes at room temperature and colonies were counted (>50).

Single Cell Gel Electrophoresis (Comet Assay)

A549 cells were seeded in 6-well plates (2 x10⁵) and treated with incubated with indicated doses of KAN0438757 for 48 h. At the end of the incubation, the wells were washed with PBS and the cells were dissociated using trypsin. The cells were then centrifuged and re-suspended with PBS and mixed with 0.5% low melting point agarose (LMA). After taking 20 μl of this mixture, it was spread on a slide covered with normal melting point agarose (NMA) and the agarose was frozen at 4 °C by covering the coverslip for 45 minutes. Coverslips were removed and placed in cold lysis solution (2.5M NaCl, 100mM EDTA, 10mM Tris, 10% DMSO, 1% Triton X-100, (pH 10)) at 4 °C for 60 min. Slides were rinsed with PBS and placed in a buffer-filled gel electrophoresis. Electrophoresis was performed at 28V, 300 mA for 25 minutes and slides were neutralized with solution (pH 7.5) and rinsed with PBS. Slides were stained with Ethidium bromide (10 µg/mL) for 15 minutes and viewed under a fluorescent microscope. Opencomet program was used for analysis.

Wound-Healing Assay

Cells were seeded in 6-well cell culture dishes at 25x10⁴ in each well. After the cells adhered to the surface, the cells were scraped to form a straight line with the help of a 20 µl pipette tip. Each well was washed twice with PBS so that the removed cells would not re-adhere to the surface. Wound areas were observed with an inverted microscope and photographed at 0, 24, and 48 hour time periods [17].

Acridine Orange And Ethidium Bromide double staining

Acridine orange and Ethidium bromide (EtBr/AO) staining was performed according to the protocol described previously [18]. A549 cells were treated with the indicated doses of KAN0438757 and then EtBr/AO(1:1) and added to the wells for 15 minutes at room temperature. Stained cells were observed with a fluorescence microscope (Olympus, Japan).

Mitochondrial membrane potential (ΔΨm) Assay

Monolayers cells in the logarithmic growth stage were inoculated into glass-bottom culture plates at 2×10⁵ cells per well. Then, cells were treated with KAN0438757 for 48h. Slides were then washed and stained with the cationic dye JC-1 (5 mg/mL) for 15 min at 37 °C. The stained cells were analyzed by fluorescence microscope (Olympus, Japan). After staining, seven areas per well were randomly captured by the microscope. Each experiment was repeated at least three times.

Protein Isolation and Western Blot Analysis

Cells were collected with a plastic scraper by washing three times with cold PBS after 48 hours of treatment and transferred to numbered microcentrifuge tubes. After centrifugation at 6600 rpm for 10 minutes, it was dissolved in lysis (RIPA) buffer and then incubated on ice for 60 minutes. After the incubation period, it was centrifuged for 10 minutes at 14,000 rpm at 4°C. Protein content in the supernatants was measured by their absorbance at 595 nm in the spectrophotometer using BSA as a standard. (Biomethod-GBC (Bradford)). With the help of electrophoresis system (Bio-RadTrans-Blot cell, BioRad, USA), protein samples and marker were fractionated on SDS-PAGE (10-12%) gel. Then, the proteins separated in the gel were transferred to Polyvinylidenedifluoride (PVDF) membrane. Blockage was made with BSA to close the pores in the membrane. The membrane was incubated overnight with primary antibodies first and then with secondary antibodies. Subsequently, washing was done with TBS-T. The results were determined by chemiluminescence method so that the membranes were visualized by X-Ray device.

Statistical Analyses

Statistical analyses were performed using the GraphPad Prism software (USA). Data were obtained from three separate experiments. Statistical analysis was analyzed with Graph Pad Prism 8.0.2 program according to multiple comparison Post-Hoc Tests in “One-way ANOVA” method or independent two-sample Student’s ttest . P < 0.05 was accepted as significant in the analyzes.

The PFKFB3 inhibitor KAN0438757 effectively blocked the proliferation of non-small cell lung carcinoma cells

Colorimetric assay (WST-1 based) and Real-Time Cell Analysis (RCTA) was carried out to investigate the effect of KAN0438757 on cell viability and the proliferation of cell lines. The Non-small cell lung cancer cell lines (NSCLC H1299 and A549) or COS7 were treated with increasing concentrations of KAN0438757 (0, 5, 10, 25, 50 and 100 µM). The WST-1 results showed that KAN0438757 inhibited cell viability in a dose-time dependent manner (supplementary data, 24/48/72 h) when compared to untreated control and the cell viability of the A549 and H1299 cell lines with KAN0438757 for 48 h were shown in Fig. 1a and b. The IC50s of A549 for KAN0438757 was pronouncedly lower (35.2 μM) than that of H1299 (52.4 μM) for 48h. RCTA was performed to further confirm the effect of KAN0438757 on the inhibition of lung cancer cells proliferation for 48h. The RCTA results of A549 cells (Fig. 1d) further proved that KAN0438757 had a stronger antiproliferative activity. Similar results also observed in H1299 cells (Fig. 1e). In addition, Colony forming assays were performed to determine if the inhibitory effects of KAN0438757 on A549 cell line proliferation are sustained over time. Consistent with the proliferation and viability results, KAN0438757 treatment also significantly inhibited colony formation (Fig. 1f).

KAN0438757 stimulate DNA damage

In order to investigate the effects of KAN0438757 on DNA damage was analyzed by Comet Assay in A549 cell line using alkaline single-cell gel electrophoresis (Fig. 2). The results demonstrate the effect of KAN0438757 after 48 h of treatment. For the lower concentration of KAN0438757, i.e. 5 μM, the level of DNA damage was incomparable, however; in 10 μM treated cells more than the control cells. The statistical analysis has demonstrated a significant effect at 25 and 50 μM. These concentrations of KAN0438757 induced a clear and significant increase in %DNA in tail and tail length (Fig. 2b and 2c).

KAN0438757 inhibits migration of A549 lung cancer cells

Furthermore, having established that a low dose of KAN0438757 has no effect on cell viability, we used a wound-healing assay in order to investigate whether these concentrations affect cell migration in the A549 cell line. The data showed that a low concentration of KAN0438757 (less than 25 μM) effectively inhibited the cell migration (Fig. 3a). The distance was found to be wide in A549 cells treated with KAN0438757 compared with the control (Fig. 3A), indicating that KAN0438757 effectively inhibits cell migration ability in A549 cells (Fig. 3b).

AO/EB double-staining in KAN0438757 treated-lung adenocarcinoma cells

After treatment with KAN0438757 inhibitors for 48 h, apoptotic cell death was determined by using Double-Staining with Acridine Orange-Ethidium Bromide on A549 lung cancer cells. As shown Fig.4a, in the untreated control group, the green color labeled cells was visible. On the other hand, KAN0438757 treated groups, the intensity of the orange color also increased, illustrating a higher rate of apoptosis, which had the highest number of apoptotic cells in the 25 μM group.

PFKFB3 inhibitor KAN0438757 triggers mitochondrial membran instability in A549 cells

The mitochondrial membrane potential (ΔΨm) is crucial for mitochondrial homeostasis which is a fundamentally important determinant of cell predestination. To investigate whether KAN0438757 induces A549 cell mitochondrial membrane instability, we used JC-1 staining which is the cationic dye; red-colored cells indicate healthy mitochondria while green-colored indicate loss of mitochondrial membrane potential. As depicted in Fig. 4b, with increasing the KAN0438757 concentration, the intensity of green color also increased, indicating a higher rate of loss of mitochondrial membrane potential in A549 cells. Furthermore, the results showed a significant positive correlation between the loss of mitochondrial membrane potential and the KAN0438757 concentration indicating that KAN0438757 triggers mitochondrial membrane instability.

KAN0438757 induces autophagy in lung adenocarcinoma cell

To further assess whether KAN0438757 affects autophagy, which are key events that maintains cellular homeostasis. To investigate the underlying mechanism of autophagy induced by KAN0438757, the expression of p62/SQSTM1, LC3 I-II, and Beclin1 was measured by western blotting. Results showed that KAN0438757 in A549 cells did not change the protein expression level of p62/SQSTM1. As shown in Fig. 4d, the protein expression of LC3 I was down-regulated and LC3 II was up-regulated in A549 cell line. Besides, KAN0438757 treatment increased the Beclin1 protein expression level. Collectively, these findings indicated that KAN0438757 treatment could induce autophagy in A549 cells.

PFKFB3 is a member of a the bifunctional enzyme family of 4 such proteins of which are known a rate-limiting enzyme and essential control point in the glycolytic pathway. Beyond its glycolytic activities, PFKFB3 appears to have functions in modulating several cell behaviors, promoting cell cycle progression(B. Clem et al. 2008) inhibiting apoptosis [19], and having a critical factor for homologous recombination repair of DNA double-strand breaks [15]. Previous studies have revealed that PFKFB3 expression is increased significantly both non-small cell lung cancer (NSCLC) [16] and small-cell lung cancer (SCLC)[20]. Gustafsson et al identified that PFKFB3 has a crucial role in homologous recombination and developed a selective PFKFB3 small molecule inhibitor- KAN0438757. However, to the best of our knowledge, there is currently no study available that describes the effects of KAN0438757 on lung cancer. Moreover, the properties of KAN0438757 regarding anti-neoplastic and induction of programmed cell death and the underlying mechanism(s) in lung cancer cells are unclear. Therefore, we assessed the effect of KAN0438757 in human lung cancer and normal cells.

In this study, we investigated the potential anti-neoplastic effect of KAN0438757 in lung cancer cell line A459 and H1299 cells, and the possible mechanisms for it by determining cell viability, cell proliferation, colony formation, DNA damage, and cell migration, the morphological analysis of apoptosis, apoptosis, and autophagy protein expression.

Firstly, the anti-proliferative effects of KAN0438757 were analyzed on A549 and H1299 cells. Similar to previous research [21], our results demonstrated that KAN0438757 effectively reduced cell viability and inhibited the growth of A549 and H1299 lung cancer cells. Furthermore, our investigation involving the effect of KAN0438757 on colony formation, demonstrated that suppressed the colony formation of A549 cells as shown by the colony formation assay for a longer period of time. Following the treatment of KAN0438757 into lung cancer cells, clonogenic survival of lung cancer cells was suppressed, while their proliferation was inhibited.

In recent years, accumulating evidence showed that PFKFB3 knockdown leads to DNA damage and inhibiting DNA repair resulting in G2/M arrest and tumor growth delay [22]. In addition, recent study reported that PFKFB3 has a crucial role in the HR repair of DNA double-strand breaks [15]. Furthermore, Ninou et al. reported that pharmacological inhibition of PFKFB3 leads to an accumulation of DNA damage in replicating cells, fork collapse, and subsequently increased genomic instability [23]. A small molecule inhibitor were also reported inhibition of PFKFB3 could lead to DNA damage and cell death. PFK15, a PFKFB3 inhibitor, cause DNA damage impairing DNA repair through AKT in HCC cells[22]. In this study, a comet assay quantitatively detected KAN0438757-induced DNA damage in A549 cells. In line with previous reports, our results showed that the Tail DNA and Tail length of the KAN0438757 treatment groups (25 and 50 µM) were higher than those of the other groups, indicating more severe DNA damage. These results indicated that KAN0438757 triggered DNA damage at the single-cell level, which may lead to apoptosis in lung cancer cell line.

One of the two main pathways that initiate apoptosis is the Mitochondria-mediated pathway, also known as the intrinsic pathway [24]. The translocation of pro-apoptotic proteins from the cytoplasm to the mitochondrial membrane triggers the disruption of mitochondrial membrane potential (Δψm) and subsequently causes the release of cytochrome-c, which will play a role in the apoptosome complex (comprising procaspase-9, APAF-1 (apoptotic protease activating factor-1), and cytochrome-c ), into the cytoplasm. The disruption of Δψm can lead to the activation of the caspase cascade including caspase-9 and caspase-3 is activated, which are crucial effectors of apoptosis [25]. Once the caspase-3 is activated, the downstream effector proteins are cleaved such as PARP, which plays essential roles in DNA repair and the programmed cell death process [26]. In the current study, it was observed that treatment with KAN0438757 induced cleavage of PARP. In addition, KAN0438757 treatment of A549 cells significantly increased the red fluorescence intensity, indicating loss of Δψm, which is an early event in the apoptotic process. Furthermore, our AO/EB dual staining data confirmed that KAN0438757 treatment increased the apoptosis at 10 and 25 µM dose of KAN0438757. These results are consistence with the former results that either siRNA silencing [19] or the pharmacological inhibition of PFKFB3 [27] induces cell apoptosis. All the results discussed above directly suggest that KAN0438757 induces A549 cell apoptosis via mitochondria-mediated pathway.

PFKFB3 also plays a role in angiogenesis and metastasis, which can indirectly promote tumor growth [28]. Previous in vivo and in vitro studies have indicated that PFKFB3 is involved in the migration in nasopharyngeal carcinoma [29], breast cancer [28], non-small-cell lung cancer [16], gastric cancer[30], and osteosarcoma Saos-2 cells [31]. In the present study, KAN0438757 was applied as an PFKFB3 inhibitor in lung cancer cell line. The results indicated that KAN0438757 significantly blocked migration of A549 cells. This result was in accordance with previous reports, where KAN0438757 was observed to reduced the migration capabilities of HCT-116 and HT-29 cell line, as well as HUVECs cells [21].

Autophagy may play different roles not only in the degradation of macromolecules and damaged organelles, but also in different steps of tumor development. It may play a role in inhibiting proliferation and metastasis of cancer cells during tumorigenesis by autophagic activity. On the other hand, as a pro-survival mechanism, cancer cells initiate autophagy by breaking down intracellular organelles and proteins, thus providing the most important energy support of cancer cells [32,33]. Previous in vitro experiments showed that either knockdown of PFKFB3 or pharmacological inhibition, including PFK158, and 3-PO, participated in autophagic flux and markers of autophagy induction [34,35]. Knockdown of PFKFB3 resulted in the degradation of p62/SQSTM1. Meanwhile, PFKFB3 siRNA transfection, led to a significant increase LC3-II which involved in the assembly of autophagosomes in the inner and outer membrane [35]. Indeed, we found that the inhibiting of PFKFB3 after KAN0438757 treatment is positively connected with autophagy in A549 cells. Western blotting results revealed that the protein expression LC3 II and Beclin1 were significantly increased in the KAN0438757 treatment, while the expression of p62/SQSTM1 was not significantly increased in A549, suggesting that the KAN0438757 treatment promotes autophagy. Accumulating evidence has shown that autophagy can promote cell survival and maintenance of cellular homeostasis by degrading damaged organelles and macromolecules in cells, but it should not be overlooked that autophagy can also promote cell death due to its tight association with apoptosis [36]. In conjunction with analysis of AO/EB and Δψm, western blotting demonstrated that KAN0438757 treatment (25 μM, 48 h) induced cell death.

The main point of our study is to first report the programmed cell death effects of KAN0438757 on lung cancer. In the present study, we found that KAN0438757 inhibited the colony formation and migration of human lung cancer cells and induced DNA damage; meanwhile, triggers mitochondrial membrane instability and subsequent induction of programmed cell death apoptosis and autophagy in the lung cancer cell. Of importance is that KAN0438757 has anti-neoplastic potential as a novel agent for therapy.

PFKFB 6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatases

PFK-1 phosphofructokinase-1

PTEN Phosphatase and tensin homolog

PI3K/Akt phosphoinositide 3-kinases

MAPK mitogen activated protein kinase

HIF-1 ahypoxia-inducible factor 1-alpha

3PO 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one

HUVECs Human endothelial cells

MMP Mitochondrial Membrane Potential

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Data availability

Data sharing does not apply to this article as no datasets were generated.

Declaration of competing interest

Authors have no conflict of interest.

Acknowledgment

This study was supported by BUBAP (Grant Numbers: BAP-FEF.2021.004 and BAP-FEF.2021.005). Deniz Özdemir and Seher Saruhan are equal contributors to this work.

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Editorial decision: Reject, do not transfer
09 Aug, 2023
Reviewers agreed at journal
19 Jul, 2023
Reviewers invited by journal
18 Jul, 2023
Editor assigned by journal
18 Jul, 2023
First submitted to journal
17 Jul, 2023

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KAN0438757: a novel PFKFB3 inhibitor that induces programmed cell death and suppresses cell migration in non-small cell lung carcinoma cells

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