Targeting the JAK1 /STAT3 Signaling Pathway: Entinostat as a Promising Treatment for Nasopharyngeal Carcinoma

doi:10.21203/rs.3.rs-4414212/v1

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Research Article

Targeting the JAK1 /STAT3 Signaling Pathway: Entinostat as a Promising Treatment for Nasopharyngeal Carcinoma

https://doi.org/10.21203/rs.3.rs-4414212/v1

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Background

In various cancer types, entinostat (MS275) inhibits histone deacetylase (HDAC) selectively and exhibits anti-tumor activity. Our study aims to investigate the effects of MS-275 on nasopharyngeal carcinoma (NPC) cells.

Methods

CNE-2 and HONE-1 cell lines were used to carry out the experiments. CCK-8, clone formation assay, DAPI staining, mitochondrial membrane potential (MMP), flow cytometry, wound healing, transwell assay and western blot analyses were used to assess MS-275's effects on NPC cells.

Results

MS-275 inhibited NPC cells from proliferating in a dose-and time-dependent manner. The G2 phase DNA proportion obviously increased with the MS-275 concentration increase (P < 0.05). MS-275 can induce apoptosis of nasopharyngeal carcinoma cells and then cause the decrease of mitochondrial membrane potential. In addition to inhibiting NPC invasion, MS-275 can also suppress cell migration. With increasing drug concentration, the phosphorylation of JAK1 and STAT3 was significantly prevented, while their expression remained unchanged. This is consistent with the immunofluorescence and confocal laser-scanning microscopy result. The metastasis-relevant MMP-2, Snail, anti-apoptotic protein Bcl-xL and Bcl-2 were all downregulated, while the metastasis-relevant E-cadherin, pro-apoptotic protein Bax and cyclin dependent kinase inhibitors p27 and p21 were significantly increased.

Conclusion

The present data demonstrates that MS-275 has the ability to induce apoptosis and suppress NPC proliferation and migration by affecting the JAK1/STAT3 signaling pathway. Therefore, MS-275 may present as a promising therapeutic agent for intractable NPC.

nasopharyngeal carcinoma

MS-275

JAK1/STAT3 signaling pathway

proliferation

apoptosis

migration

The chemical compound entinostat which is generically known as MS-275 is one of the benzamide class HDACIs. Histone deacetylases (HDAC) is a kind of protease that play an imperative role in chromosome structural modification and gene regulation. In general, HDACs promote the dissociation between DNA and the histone octamer, leading to relaxation of the nucleosome structure. This process facilitates various gene transcriptions when transcription factors and co-transcription factors bind to DNA binding sites. The histone acetylation and deacetylation are in dynamic equilibrium in the nucleus, which are regulated by histone acetyltransferase (HAT) and HDAC. HAT transfers the acetyl of acetyl coenzyme A to a specific lysine residue in histone tails. HDAC induces histone deacetylation, rather than tightly binding with a negative charged DNA. This results in chromatin dense curled up, and the suppression of gene transcription.

The excessive expression of HDAC results in an increase in acetylation in cancer cells. Restoring the positive charge of histones can enhance the interaction between DNA and histones, causing the nucleosome to become tightly packed. This compacted structure is unfavorable for the expression of specific genes, including tumor suppressor genes. HDAC inhibitors (HDACIs) are compounds that disrupt the function of HDAC. They can promote histone acetylation in specific chromatin regions, thereby regulating apoptosis, protein expression and stability, such as increasing the expression of the p53 and p21 genes, inhibiting tumor cell proliferation, and inducing cell differentiation and/or apoptosis ^[1–2]. HDACIs have emerged as a novel class of drugs that demonstrate therapeutic efficacy in various central nervous system diseases and cancers ^[3], They offer the advantages of high selectivity and low toxicity to tumor cells. The anti-metastatic, anti-invasive, anti-migratory, and anti-angiogenic effects of HDACIs have been confirmed. Moreover, they have shown anti-metabolic and anti-angiogenesis activities in vitro and in vivo ^[4–5]. HDACIs have become a major focus in cancer therapy research. For instance, they can upregulate the expression of glycoprotein RECK, thereby downregulating matrix metalloproteinases and inhibiting tumor metastasis and angiogenesis ^[6].

Since the 1990s, a variety of different structures ofHistone deacetylases inhibiters (HDACIs)chemical compounds have been obtained, and a significant amount of HDACIs have given access to the stage of clinical trials. Entinostat (MS-275) one of the benzamide class HDACIs could strongly inhibit HDAC1 and HDACI3. In a recent study, researchers have found that MS-275 can inhibit various kinds of cancer cell proliferation, such as gastric cancer, cervical cancer, liver cancer, lung cancer, osteosarcoma, neuroblastoma, medulloblast, retinoblastoma, and rod malignancies ^[3]. However, there are few studies on nasopharyngeal carcinoma (NPC) ^[7]. On the basis of a previous study, our study aims to find out the effect of MS-275 in two poorly differentiated NPC cell lines, and verify the anti-NPC influence of MS-275, for the better future therapy of NPC.

Materials

MS275 was purchased from Sigma Aldrich (lot number: 4K01159) and dissolved in dimethyl sulfoxide (DMSO) at a storage concentration of 10 mM. It wsa kept at -20˚C until use. DAPI (Cat.C1005) and Hochest33258 (Cat.C1018) were obtained from Beyotime Institute of Biotechnology (Haimen, Jiangsu, China). The cell counting kit-8 (Cat.C008-3) was purchased from 7sea biotech (Baoshan, Shanghai, China). The apoptosis detection kit (Cat.A211) and cell cycle assay kit (Cat.A411) were supplied by Vazyme (Nanjing, Jiangsu, China). The second antibodies were bought from Thermo Fisher (Waltham, MA, USA). The primary antibody for STAT3 was obtained from Santa Cruz biotechnology (Delaware, CA, USA), while the primary antibodies for β-actin, p-stat3, MMP2, snail, e-cadherin, Caspase-3, Bcl-XL, Bax, Bcl-2, as well as P21 and P27 were supplied by Cell Signaling Technology, Inc. (Danvers, MA, USA). Our articles have been reviewed and edited by professional native speakers.

Cell culture

The HONE-1 and CNE-2 cell lines, which are two human nasopharyngeal carcinoma cell lines, were provided by the Faculty of Medicine, Chinese University of Hong Kong. The cells were cultured using RPMI 1640 medium (Gibco) supplemented with 100 µg/mL penicillin-streptomycin (Beyotime Biotech, Haimen, China) and 10% fetal bovine serum (Gibco). The cells were incubated at 37˚C with 5% CO2.

CCK-8 assay detecting cell proliferation

Using the CCK-8 assay to determine the effect of MS-275on cell viability/proliferation. Briefly, 5000 CNE-2 or HONE-1 cells were seeded in 96-well plates and cultured for one day. The NPC cells were then treated with various concentrations of MS-275 (0, 1.25, 2.5, 5, 10, and 15 µM) for 0, 12, 24, 48, 72, or 96 hours. Cell proliferation was assessed using a CCK-8 kit following the manufacturer's instructions. All the tests were repeated three times. The absorbance of the samples at 450 nm (with a reference at 655 nm) was measured using a Bio-Rad iMark (Bio-Rad, Hercules, CA, USA). The inhibition of MS-275 in NPC cells was calculated, and the IC50 of MS-275 in NPC cells was analyzed using GraphPad Prism 5.0 software.

Colony formation

The frequency of HONE-1 and CNE-2 cells was determined using the colony formation assay. The cells were treated for 24 hours with MS-275 of various concentrations (0, 2.5, 5, 10µM) in 6 cm plates. Alternatively, we plated cells at a density of 700 cells in culture plate filled with complete medium and then we incubated the plate in a 5% CO₂ incubator. After an incubation period of 10 days, the cells were subjected to two washes with cold PBS and then fixed with cold formaldehyde for 15 minutes. Following fixation, crystal violet staining was conducted for 5 minutes, followed by three subsequent rinses with distilled water. The cells were allowed to air dry, and subsequently, the colony forming ability was determined.

Wound healing assay

First sweeping out the "一" glyph mark on the back of the 6-well plate with a ruler for determining the follow-up scratch position. Briefly, HONE-1 and CNE-2 cells were seeded at a density of 3×105 cells per well in 6-well plates. The cells were then gently agitated and evenly distributed using the crisscross method to ensure uniform growth until confluence was attained. A sterile 20µl micropipette tip was employed to create a consistent wound by scratching the cell monolayer. After three washes with PBS, images of the scratches, including the adjacent leading edges of cells (˟100 magnification), were captured. Subsequently, 1 ml of RPMI1640 culture medium containing various concentrations of entinosta (0, 2.5, 5, 10µM) was added to each well. The rate of wound closure was monitored using a phase-contrast microscope and documented through image capture. This experiment was repeated three times, with six scratches evaluated during.

Migration

The logarithmic phase cells with good condition were starved for 24 hours in serum-free medium (conditioned medium), then using the trypsin to digest the cells and finally collecting the cells when digesting finish. The cells resuspended in conditioned medium with a proper concentration of 5x10⁴/ml. Adding 1µl of drug storage solution to 1 ml cell suspension to dilute the drug content to 10 µM, then the cell suspension was diluted by multiple ratios to obtain working solution which contain different concentrations MS-275 (0, 2.5, 5, 10µM). The cell suspension 100µl was seeded onto the upper chamber insert and put in a 24-well plate that contain 650µl complete medium. Intervention in a 37℃, 5% CO² incubator for 48 hours, the old culture medium was aspirated, using the cotton swabs removing the non-migrating cells, and using PBS washing it twice, fixing with 4% paraformaldehyde for 5min and washing one more time. Adding 650µl crystal violet stain into the lower chamber, which could cover the chamber membrane completely, and incubating at room temperature for 30min. In the lower chamber, another 650µl of crystal violet stain was added and incubated at room temperature for 30 minutes, followed by three washes with PBS. The number of cells that passed through the membrane of the chamber was observed under 200× magnification (×10, ×20) and were either photographed or counted in five random views.

Cell cycle analysis

Collecting the HONE-1and CNE-2 cells after treating the NPC cells with MS-275 (0, 2.5, 5, 10µM) for 48h and fixing them with 75% cold ethanol for one hour, and then storage at -20°C overnight. Subsequently, the cells were washed twice with cold PBS (pH 7.4) and stained with PI staining solution following the manufacturer's protocol. Analyzing the cell cycle distribution was by flow cytometry (Beckman, MCL, USA) and the distribution result was analyzed by Cellquest software.

Apoptosis analysis

In order to measure the apoptosis of HONE-1 and CNE-2 cells, we choose an apoptosis detecting kit (Vazyme biotech, China). Briefly, seeding these two cell lines in 6-well plates respectively and incubating 36h. Then the cells were treated MS-275 (0, 2.5, 5, 10µM) for 48h.Then using pancreatin without EDTA digest the cells and collecting the cells. Washing the cells with PBS twice and the cells were stained separately with Annexin V-FITC and PIon the basis of the instructions. The fluorescence results were measured by the cellquest analysis software in a flow cytometer (Beckman, MCL, USA).

DAPI staining

About 3 × 10⁵ HONE-1and CNE-2 cells in 6-well cell culture plates overnight. After that incubating them with MS-275 (0, 2.5, 5, 10µM) for 48h. When the incubation was finish, washing the cells with ice cold PBS and then fixing the cells with methanol for 15min, staining the cells with 500µl DAPI dye liquor for half an hour away from light at room temperature. At last, washing the samples thrice with PBS and capturing the sample images by the fluorescence microscope (Nikon, Japan).

Measurement of mitochondrial membrane potential (MMP)

A density of 2 × 10⁵/well logarithmic phase CNE-2 and HONE-1 cells were seeded in 6-well plates and treated with or without MS-275 (0, 2.5, 5, 10 µM) for 48 h. The old medium was discarded, and 1 mL of jc-1 staining solution was added to the cells. After thorough mixing, the cells were incubated at 37℃ for 20 minutes in a cell culture box. Meanwhile, 4 mL of distilled water was added to 1 mL of jc-1 staining buffer (5X), and the resulting volume was made up to obtain a 1X jc-1 staining buffer, which was incubated on ice. After the 20-min 37℃ incubation was finish, absorbing the supernatant, using the cold jc-1 staining buffer (1X) wash twice, and then adding 2ml cell culture medium and the changing fluorescent intensity was measured using fluorescence microscopy (Binocular inverted biological microscope, Nikon, Japan).

Western blotting

The NPC cells were washed thrice by ice-cold PBS. Preparing the cell lysates by using the RIPA buffer (Beyotime Biotech, haimen, China) that contain 1% protein inhibitors which include Phenylmethanesulfonyl fluoride (PMSF) and protein phosphatase inhibitor. After a 10-seconds forced vortex, incubating the cell lysates on ice for 30 mins, during the ice incubation, the cell suspension was blown every five minutes with a small tip as to fully lyse the cells and then centrifuging the cell lysates15 min with 12,000 rpm at 4˚C. Transferring the supernatant to a 1.5 mL dry clean EP tube. Using the BCA method (Beyotime Biotech, haimen, China) to measure the protein concentrations. Boiling the cell supernatant for 10 mins which was add a ratio of Loading Buffer (5×) and last the buffer volume was diluted to 1×. After electrophoresis in SDS-PAGE, transferring the band onto a PVDF membranes (Immobilon-P, Millipore, USA). Blocking the membrane with 2h in 5% skim milk at room temperature. Incubating the membranes at 4℃ overnight by using specific primary antibodies then using the enzyme-linked secondary antibodies to incubate the membranes

for 1h at 25℃. Then visualizing the results by enhancing chemiluminescence (ECL, Bio-Rad, USA), Using the ChemiDoc XRS transilluminator (Bio-Rad, USA) to analyze the result. Image output and editing, grayscale scanning was measured by Image J Gray Analysis soft.

Statistical analysis

Each test was repeated at least 3 times and three parallel wells were set for each concentration group. Using SPSS19.0 to analyzed data: Measurement data were reported by Mean ± SD (\(\stackrel{-}{x}\) ± s). Multiple groups test results, multiple variables and multiple time points were measured by repeated ANOVA. Pairwise comparison among multiple groups was performed by ANOVA. Homogeneous variance was tested by LSD, and heterogeneous variance was tested by Dunnett's T3. A p-value of less than 0.05 or 0.01 for each treating group versus control was visible to show statistically significant.

MS-275 shows high inhibition in CNE-2 and HONE-1 cells

The effects of MS-275 on the proliferation of CNE-2 and HONE-1 cells were initially carried out. As presented in Figs. 1A and 1E, MS-275 could significantly inhibit the growth of CNE-2 and HONE-1 cells in a dose- and time-dependent manner. From the results we can see that the inhibition rate of 5 µM MS-275 in HONE-1 and CNE-2 cells were 10.07%, 17.74%, 31.30%, 57.65% and 69.97%, and 13.36%, 27.35%, 57.18%, 80.94% and 92.85% after treatment for 12, 24, 48, 72 and 96 hours, respectively. The rate of inhibition reached 83.36% and 91.87%, respectively, after treatment 15 µM MS-275 for 96 hours. Curve fitting was employed to analyze the IC50 values at different time points (Figs. 1B and 1F). Among the selected time points (12, 24, 48, 72, and 96 hours), the IC50 values at 48 hours were found to be 8.84 µM and 3.90 µM for the respective cell lines. Based on the IC50 results, experiments were conducted using 2.5, 5.0, and 10.0 µM MS-275 for 48 hours. In addition, the colony formation assay verified the suppressive effect on the proliferation of MS-275. A decrease in colony formation was observed, which is characterized by the number and volume of cells. Compared to untreated cells, MS-275 treated cells formed smaller colonies in a dose-dependent manner (Figs. 1C and 1G). The colony formation of CNE-2 and HONE-1 cells were suppressed by MS-275 (P < 0.05, Figs. 1D and 1H). And the suppression of MS-275 was enhanced with the increase of its concentration. Overall, the results indicate that MS-275 inhibited the proliferation and growth of NPC HONE-1 and CNE-2 cells effectively.

Cell cycle arrest in NPC cells after treating MS-275

The effect of MS-275 on the cell cycle of HONE-1 and CNE-2 cells was observed. These two cell lines were treated with the same method as before. The results depicted in Figs. 2A-2D revealed a significant accumulation of cells at the G2/M phase (P < 0.05) in both cell lines, while CNE-2 cells were also arrested at the S phase (P < 0.05) in an MS-275 concentration-dependent manner. In summary, these findings indicate that MS-275 can cause cell cycle arrest in NPC cells, and affect cell DNA replication. It is noteworthy that a sub‑G1 apoptotic peak could be observed in the 5-µM CNE-2 group and 10-µM HONE-1 group. This shows the different concentration effects of MS-275 in these two cell lines.

Suppression of the migration of NPC cells

MS-275 relatively low toxicity to cells and effectively inhibits cell migration. The experimental results in Fig. 3A demonstrated this point. After the 48-hour treatment, the 10-µM concentration group exhibited a stronger inhibition of migration compared to the control, 2.5-µM and 5-µM concentration groups (Fig. 3B). The Boyden chambers coated without collagen confirms the migration inhibition of MS-275 to CNE-2 and HONE-1cells. As shown in Fig. 3C, with the increasing concentrations of MS-275, the migration of cells gradually decreased. This confirms that MS-275 effectively inhibits cell migration (P < 0.001). It is obvious that among all the concentrations groups, the 10-µM concentration group exhibited the best effect in suppressing cell migration.

MS-275 induced apoptosis in CNE-2 and HONE-1cells

Further studies were conducted on the anti-tumor effect of MS-275 on NPC cells, which was mainly conducted by detecting the induction of apoptosis. Apoptosis is a kind of programmed cell death. When apoptosis occurs, the nucleus undergoes specific morphological changes, including nuclear aggregation, nuclear fragmentation, and the production of apoptotic bodies. As shown in Fig. 4, MS-275-treated CNE-2 cells induced obvious apoptotic morphological changes and apoptotic bodies, especially in the high-concentration group (10 µM). The HONE-1 nucleus revealed obvious chromatin condensation and concentration after DAPI staining, but no obvious apoptotic corpuscles were observed.

Next, we employed an apoptosis detection kit to investigate whether MS-275 could induce apoptosis in HONE-1 and CNE-2 cells. To detect phosphoryl serine (PS) on the cell membrane surface during apoptosis, we utilized FITC-labeled recombinant Annexin V. PS is mainly distributed in the inner membrane. At the early stage of cell apoptosis, different types of cells could roll over PS to the outside of cells and bind with Annexin V. Figure 4B demonstrates the green fluorescence of FITC-labeled Annexin V, and flow cytometry was employed to measure the percentage of apoptotic cells. The results are presented in the lower right corner E4. After treating cells with MS-275 for 48 hours, the apoptosis rate of both CNE-2and HONE-1 cells significantly increased with the escalating drug concentration. A hallmark of early apoptosis is the decline in mitochondrial membrane potential (MMP). JC-1, a commonly used detection method, allows for easy identification of this decline by observing the change in fluorescence from red to green. As depicted in Fig. 4C, upon treatment with MS-275, the distinct red fluorescence diminished in both CNE-2 and HONE-1 cells, while the green fluorescence gradually intensified with increasing concentrations of MS-275, indicating apoptosis in both cell lines.

MS-275 can reduce the expression of p-STAT3

The morphological comparison revealed that p-STAT3 was widely distributed in the cytoplasm and nucleus of NPC cells of the control group. The localization of p-STAT3 protein cells remained unchanged, but the red fluorescence was significantly reduced in the MS-275 treatment group (Fig. 5A). From the molecular expression level, and as shown in Fig. 5B, MS-275 could depress the expression of p-JAK1 to prevent p-STAT3 from transferring into the cell nucleus to regulate downstream genes expression in a dose-dependent manner. These two different detected levels revealed relevant results to each other, which indicating a potential anti-cancer pathway of MS-275 in NPC cells. Various cytokines receptors can activate the signal transducer and activator of transcription (STAT) protein family, which serves as a carrier in the interaction process between cytokine receptors and maintains the inherent specificity of signal transfer within a cell. When cells were treated with different concentrations of MS-275, the JAK1 protein became phosphorylated, and STAT3 became rapidly phosphorylated. After STAT3 and its receptor bounded together, the 705th of the C-terminus tyrosine phosphorylation (Y705) was activated, and was phosphorylated by JAK1, that is, p-STAT3. The p-STAT3, through its SH2 area, makes itself form the same type or different dimers, and transfer to the nucleus to interact with other proteins that have a particular gene promoter region of DNA elements, or other transcription factors or subsidiaries. Then, the interaction complex regulates the downstream target gene transcription and expression, including Bcl-2, Bcl-xL, Fas, Mcl-1, cyclin D1, surviving, c-myc and others ^[8]. Such regulation can induce cell proliferation, inhibit cell apoptosis, and lead to cell malignant transformation and tumorigenesis ^[9].

Changes of downstream p-STAT3 genes

(1) MS-275 increased P21 and P27 in NPC cells

Figure 5C demonstrates a dose-dependent upregulation of P21 and P27 expression after 48 hours of MS-275 treatment. The high expression of P21 and P27 suggests that NPC cells can enhance the sensitivity of anticancer factors under the effect of MS-275, thus prompting NPC cells start their own anti-cancer grow gene expression program, so as to inhibit tumor growth. For example, P27 can participate in cell cycle regulation with various ways, ultimately inhibiting cell division and proliferation, and promoting cell differentiation and regulation. Recent studies ^[10] have increasingly demonstrated that a dysregulated cell cycle is a significant factor contributing to the malignant transformation of cell proliferation. P21 and P27 are cell cycle-dependent protein kinase inhibitors, and are regarded as two candidate tumor suppressor genes that are crucial for regulating the cell cycle. Both of these belong to cyclin-dependent kinase inhibitors (CDKIs) that negatively regulate the cell cycle. Therefore, the abnormal expression of P21 and P27 is linked to the onset and progression of tumors.

(2) MS-275 changed the expression of E-cadherin, Snail and matrix metalloproteinase 2 (MMP2) in NPC cells

Figure 5D demonstrates that increasing the concentration of MS-275 leads to a significant increase in E-cadherin protein expression, while causing a significant decrease in MMP-2 protein expression in CNE-2 and HONE-1 cells. The high concentration group exhibited a significant decrease in Snail protein expression, whereas no significant changes were observed between the medium and low-concentration groups. These data suggest that MS-275 might inhibit CNE-2 and HONE-1 cell epithelial-mesenchymal transition (EMT)by downregulating Snail and MMP2, and upregulating E-cadherin. Invasion and metastasis are the most important biological characteristics of malignant tumors. EMT is a change process that refers to specific physiological or pathological conditions, with the polarity of epithelial cell changes into mesenchymal characteristic cells, and this is closely correlated to the invasion and metastasis of malignant tumors ^[11]. Through the regulation of a number of the downstream genes, such as Snail and Twist, STAT3 can play an important role in the EMT. Zinc finger protein transcription factor Snail, as a key regulating factor in the phenomenon of EMT ^[12], can downregulate the epithelial E-cadherin to decrease cell adhesion ability, and upregulate the expression of MMP2 to increase the aggressivity of tumor cells to promote tumor metastasis.

(3) MS-275 downregulated the expression of Bcl-2 and Bcl-XL, and upregulated the expression of Bax and caspase-3.

The protein expression levels of Bcl-XL, Bcl-2, Bax, and caspase-3 play a crucial role in the regulation of cell apoptosis ^[13–14]. Following treatment of NPC cells with MS-275, western blot analysis was conducted to assess the levels of these proteins in CNE-2 and HONE-1 cells. The representative immunoblotting results for CNE-2 and HONE-1 cells are depicted in Fig. 5E. The findings illustrate that treatment with MS-275 at concentrations of 2.5, 5.0, and 10.0 µM for 48 hours led to a dose-dependent reduction in the expression of the anti-apoptotic factors Bcl-2 and Bcl-XL, along with an increase in the expression of the pro-apoptotic factor Bax and caspase-3 in both cell lines. These results suggest that MS-275 can induce apoptosis in CNE-2 and HONE-1 cells by downregulating the expression of Bcl-2 and Bcl-XL, while upregulating the expression of Bax and caspase-3. The upward trend in the Bcl-2/Bax ratio, along with the upregulation of caspase-3 expression, indicates the apoptotic induction of MS275 in NPC cells.

Due to the special anatomical location and pathological type, approximately 70% of NPC patients have advanced stage Ⅲ and Ⅳa. Synchronized radiotherapy and chemotherapy, which are the main treatments for locally advanced NPC, can achieve certain curative effects. However, chemotherapy drugs often have side effects that adversely affect patients' quality of life, and the characteristics of local recurrence and distant metastasis make the treatment of locally advanced NPC often fail to keep the patient alive. Therefore, it is necessary to develop low toxicity and high efficiency therapies that can inhibit the distant metastasis of tumors.

MS-27 can selectively inhibit class I histone deacetylase. Studies have shown that MS-275 can inhibit many solid tumors and hematological tumors in vitro and in vivo, but has no significant effect on normal cells ^[15]. In TRAIL-resistant breast cancer cells, MS-275 can sensitize the apoptotic effect of TRAIL, inhibit angiogenesis, inhibit migration and invasion, and reverse the EMT ^[16]. Furthermore, MS-275 can induce the expression of the p21WAF-1/CIP-1 gene and block the cell cycle of tumors, independent of the activity of p53 ^[17]. The occurrence and development of NPC are closely related to epigenetic abnormalities. MS-275 is an important epigenetic regulatory drug, and it needs to be validated whether this can play an effective role in the treatment of NPC. Our study explored the role of MS-275 on two poorly differentiated NPC cells, CNE-2 and HONE-1, in which HONE-1 contains the Epstein-Barr (EB) virus. Cell Counting Kit-8 (CCK-8) and clone formation experiments revealed a time- and dose-dependent inhibitory effect of MS-275 on the proliferation of NPC cells, without significant differences observed between the two cell lines. Furthermore, it was confirmed that MS-275 can inhibit the proliferation of individual cells. A complete cell cycle consists of five phases: G0 (quiescent phase), G1 (prophase of DNA synthesis), S (anaphase of DNA synthesis), G2 (anaphase of DNA synthesis), and M (phase of DNA synthesis). The normal operation of a cycle requires three cycle checkpoints, namely, G1→ S, G2→ M, and a spindle checkpoint that can respond to different stimuli in different manners, and coordinate cell proliferation and death. Cell cycle destruction is caused by the inactivation of cell cycle checkpoint mutations, which is a common feature of almost all tumors. Previous studies have shown that MS-275 can block lung cancer and multiple myeloma cells at the G2 and S stages by activating p21WAF1/CIP1 in CDKIs, thereby inducing anti-tumor effects ^[18]. In our study, treatment of NPC cells with MS-275 for 48 hours led to a significant increase in the proportion of G2 cells in the high-concentration drug group compared to the low-concentration drug group or control group, indicating cell cycle arrest at the G2 phase and the induction of apoptosis in NPC cells. DAPI staining, flow cytometry Annexin V-FITC, and mitochondrial membrane potential assay revealed a significant increase in apoptotic rates of CNE-2 and HONE-1 cells with increasing concentrations of MS-275. Unlike necrosis, apoptosis is a process of active cell death regulated by genes. Morphological characteristics of apoptosis include reduced cell volume, formation of plasma membrane vesicles, cytoplasmic condensation, nuclear chromatin condensation, and ultimately the formation of apoptotic bodies that are subsequently phagocytosed ^[19]. In the early stage of apoptosis, the mitochondrial permeability transition pore (MPTP) ^[20] opens excessively under the stimulation of a specific apoptotic signal, increases mitochondrial membrane permeability, and disintegrates and decreases the transmembrane potential, which is the key event of the mitochondrial apoptotic pathway.

Invasion and metastasis are important biological characteristics of malignant tumors, which are affected by inflammatory factors, growth factors, target organ secretory proteins, metabolites, extracellular matrix enzymes and other factors. The main processes of metastasis include cell adhesion to basement membrane, the degradation of the extracellular matrix, cell movement and invasion, and the formation of metastatic foci. Numerous studies have indicated that NPC is prone to distant metastasis, but the mechanism of metastasis remains unclear. The scratch test and Trans well chamber results revealed that MS-275 can significantly inhibit the mobility and migration of NPC cells, thereby regulating both local invasion and distant metastasis of tumor cells. Schech et al. ^[21] assessed the occurrence, development and metastasis of breast cancer by bioluminescence imaging. The research result reveal that MS-275 could not only significantly control the growth of breast cancer xenografts, but also inhibit the lung metastasis in mice.

Based on the remarkable activity of MS-275 in NPC cells, the mechanism of its action on NPC cells was subsequently explored. The JAK1 protein became phosphorylated, and STAT3 became rapidly phosphorylated. STAT3 is located on chromosome 17 Q (21,31), which can be activated by ligands, such as the epidermal growth factor, binding with the DNA to regulate the transcription of target genes. STAT3 is activated when the serine (S727) or tyrosine (Y705) in its transcriptional activation domain is phosphorylated. Phosphorylation of JAK1 leads to phosphorylation of tyrosine (Tyr705) in STAT3, resulting in the activation of STAT3. Activated p-STAT3 would produce dimerization, transfer to the nucleus, and bind to DNA, in order to regulate the transcription of target genes, ultimately influencing cell proliferation, differentiation and apoptosis ^[22]. The inhibition of the STAT3-mediated signaling pathway can inhibit tumor cell proliferation, the induction of apoptosis, and the hindrance of the angiogenesis and migration of tumor cells. Therefore, the development of STAT3 inhibitors targeting STAT3 has attracted much attention in recent year. The STAT3 gene is ubiquitous in human malignant tumors. It has been reported that STAT3 is activated in more than 75% of NPC tissues ^[23]. Referring to the literature, MS-275 has demonstrated the ability to inhibit the STAT3 signaling pathway in various types of cancer cells. In conclusion, this study integrates the STAT3 signaling pathway into the molecular mechanism of MS-275 in inhibiting NPC cells. The content of total STAT3 and activated STAT3 (i.e. p-STAT3 [Tyr705]) protein in NPC cells treated with MS-275 for 48 hours was detected by immunoblotting and confocal immunofluorescence staining. As the concentration of MS-275 increased, the levels of p-STAT3 protein inside and outside the nucleus significantly decreased, while the total protein content of STAT3 did not significantly change, suggesting that MS-275 can inhibit the activation of STAT3 and block the STAT3-mediated signal pathway in NPC cells.

P21, a well-known tumor suppressor gene, plays a role in impeding the cell cycle at the G1/S or G2/M phase by inhibiting CDK activity, consequently influencing cancer cell proliferation. Studies have shown that tumor cells with a high expression of p21waf1/cip1 are significantly less susceptible to HDACi-induced apoptosis ^[24]. MS-275, as a kind of highly active HDACI, can upregulate the expression of p21waf1/cip1, and block the cell cycle of various kinds of tumors. Cumulative experiments have found that CyclinD1, CyclinE and p21 are STAT3 target genes. After blocking the STAT3 signaling pathway, the levels of P21 and P27 protein increased, and the cell cycle of B cell line BAF/B03 was blocked in the G1 phase. Our study results showed that MS-275 elevated the expression of both P21 and P27 proteins, leading to arrest in the G2/M phase of NPC CNE-2 and HONE-1 cells. The results are basically consistent with the previous research. P21 and P27 are important members of the CKI family. P21 inhibits the activities of CDK2, CDK4 and CDK6, and p27 inhibits the complex of CyclinD1/CDK4 and CyclinE/CDK2, thereby blocking the cell cycle. Therefore, it was speculated that MS-275 inhibits STAT3 signaling, upregulates the expression of p21 and p27 genes downstream, and induces cell cycle arrest in the G2/M phase.

At present, apoptosis occurs via three main pathways: death receptors, endoplasmic reticulum, and endogenous mitochondrial pathways. These pathways of cell apoptosis are not completely independent, and can be interrelated in some cases. The endogenous mitochondrial pathway is the most classical of the three signal transduction mechanisms. Bcl-2 protein family mainly regulates mitochondrial pathway apoptosis, and through intracellular displacement, this controls the inner and outer permeability, and membrane potential, which directly participates in the activation and initiation of intracellular apoptosis ^[25]. Various studies have shown that HDACi can activate the cancer cells mitochondrial apoptotic pathway by regulating the protein expression of Bcl-2 protein family which consists of three main types: apoptotic proteins (Bax and Bak), anti-apoptotic proteins (Bcl-xL and Bcl-2), and BH3-only proteins (Bid, Bim, etc.). Bax plays a key role as the primary mediator in the mitochondrial apoptotic pathway ^[26]. Initially dispersed in the cytoplasm, activated Bax translocates into the mitochondria and interacts with the outer mitochondrial membrane's permeability transition pore (mPTP). Consequently, this disruption leads to the destruction of membrane permeability and the release of Cytochrome c (Cytc), ultimately promoting the mitochondrial apoptosis pathway. The anti-apoptotic proteins Bcl-2 and Bcl-xL counteract the action of apoptotic proteins, such as Bax, by inhibiting the release of Cytc. The ratio of Bcl-2/Bax determines the occurrence of apoptosis. As the Bax protein ratio increases, the mitochondrial membrane permeability decreases, facilitating the release of apoptotic factor Cytc within the mitochondria, which in turn activates caspase-3. Caspase-3 is then cleaved from its precursor form to its activated form, initiating a series of downstream apoptotic processes ^[27]. Jona et al. ^[28] reported the inhibitory effects of MS-275 on Hodgkin's lymphoma cell proliferation, with stronger inhibition observed with higher concentrations and longer exposure times. MS-275 upregulated cyclin P21, induced apoptosis via endogenous pathways, and downregulated XIAP as well as anti-apoptotic proteins Bcl-xL and Bcl-2. However, it did not influence the expression of Mcl-2 and Bax. Similarly, Edwards et al. ^[29] demonstrated that treatment of lung cancer cells expressing high levels of epidermal growth factor receptor (EGFR) with the deacetylase inhibitor LBH-589 induced apoptosis by acetylating heat shock protein 90 (Hsp90), leading to the inhibition of the EGFR/STAT3 signaling pathway and the regulation of downstream bcl-2 44 protein family (bcl-xl, bcl-2, and Mcl-1) expression. In the current study, NPC cells treated with MS-275 for 48 hours exhibited increased expression of Bax, inhibition of Bcl-2 and Bcl-xL, as well as reduced levels of caspase-3 precursor. This was consistent with the effect of MS-275 and other HDACi on the expression of apoptotic-related factors in various malignant tumors. These present results suggest that MS-275 can alter the permeability of the mitochondrial membrane by modulating the Bax/Bcl-2 ratio, leading to the release of Cytochrome C and activating the procaspase-3, which can then be cleaved into its active form, caspase-3, ultimately inducing apoptosis. In summary, this study reveals the mechanism of MS-275 in inducing apoptosis in NPC cells through the classical mitochondrial pathway, in order to exert its inducing effect.

The lack of intercellular junction is an important link in tumor metastasis and invasion. The expression of matrix metalloproteinases (MMPs) would be upregulated after tumor cells detach from the primary lesion and enters the stroma. Among these MMPs, MMP-2 is one of the hottest proteases, and is also one of the most important downstream proteins of the STAT3 signaling pathway. This can effectively degrade collagen Ⅳ, Ⅴ and laminin, which has close associate with invasion, diffusion and metastasis of tumors ^[11]. E-cadherin, a cell adhesion molecule, plays a vital role in tumor development, invasion, and metastasis. Downregulating or silencing the expression of E-cadherin induces EMT. Previous studies have indicated that E-cadherin protein can predict and influence the sensitivity of gefitinib treatment in non-small cell lung cancer. Pretreatment with MS-275 enhances the expression of E-cadherin protein and augments the effect of gefitinib treatment ^[30]. Snail, a member of the zinc finger protein superfamily, promotes cell metastasis by binding to E-box elements on the promoter of E-cadherin, thereby inhibiting its transcription. This adhesion molecule of epithelial cells eventually leads to EMT ^[17]. Snail exhibits high expression in various solid tumors and is considered an important factor in promoting tumor metastasis and invasion. In this study, based on preliminary epigenetic research, three metastasis-related proteins, Snail, E-cadherin, and MMP-2, were assessed at the molecular biology level. Some of these results demonstrated that MS-275 decreases the expression of Snail and MMP-2 in NPC cells, enhances the expression of E-cadherin, and decreases cell migration ability. The high or low expression of these factors indicates that MS-275 can inhibit the metastasis and invasion of NPC cells. However, further studies are required to elucidate the specific molecular mechanism.

MS-275 may regulate the expression of related proteins via inhibition the JAK1/STAT3 signaling pathway (Fig. 6), specifically, by upregulating the expression of cyclic inhibitory proteins P21 and P27, leading to cell cycle arrest in the G2/M phase in CNE-2 and HONE-1 cells. Furthermore, through mitochondrial apoptotic pathway, it reduces the Bcl-2/Bax protein ratio, and induces NPC cell apoptosis. In addition, through regulating the expression of metastasis-related proteins Snail, E-Cadherin and MMP-2, it inhibits NPC cell metastasis. Therefore, MS-275 may be a new therapeutic method for patients with locally advanced or metastatic NPC.

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The authors declare that they have no competing interests.

Funding

Medical Scientific Research Foundation of Guangdong Province grant (no. A2018520).

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Targeting the JAK1 /STAT3 Signaling Pathway: Entinostat as a Promising Treatment for Nasopharyngeal Carcinoma

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Abstract

Background

Methods

Results

Conclusion

Figures

Background

Materials and Methods

Materials

Cell culture

CCK-8 assay detecting cell proliferation

Colony formation

Wound healing assay

Migration

Cell cycle analysis

Apoptosis analysis

DAPI staining

Measurement of mitochondrial membrane potential (MMP)

Western blotting

Statistical analysis

Results

MS-275 shows high inhibition in CNE-2 and HONE-1 cells

Cell cycle arrest in NPC cells after treating MS-275

Suppression of the migration of NPC cells

MS-275 induced apoptosis in CNE-2 and HONE-1cells

MS-275 can reduce the expression of p-STAT3

Changes of downstream p-STAT3 genes

(1) MS-275 increased P21 and P27 in NPC cells

Discussion

Conclusion

Declarations

References

Additional Declarations

Status:

Version 1