A new triazolothiadiazine derivative inhibits stemness and induces cell death in HCC by oxidative stress dependent JNK pathway activation

Hepatocellular carcinoma (HCC) is a highly heterogeneous cancer, and resistant to both conventional and targeted chemotherapy. Recently, nonsteroidal anti-inflammatory drugs (NSAIDs) have been shown to decrease the incidence and mortality of different types of cancers. Here, we investigated the cellular bioactivities of a series of triazolothiadiazine derivatives on HCC, which have been previously reported as potent analgesic/anti-inflammatory compounds. From the initially tested 32 triazolothiadiazine NSAID derivatives, 3 compounds were selected based on their IC50 values for further molecular assays on 9 different HCC cell lines. 7b, which was the most potent compound, induced G2/M phase cell cycle arrest and apoptosis in HCC cells. Cell death was due to oxidative stress-induced JNK protein activation, which involved the dynamic involvement of ASK1, MKK7, and c-Jun proteins. Moreover, 7b treated nude mice had a significantly decreased tumor volume and prolonged disease-free survival. 7b also inhibited the migration of HCC cells and enrichment of liver cancer stem cells (LCSCs) alone or in combination with sorafenib. With its ability to act on proliferation, stemness and the migration of HCC cells, 7b can be considered for the therapeutics of HCC, which has an increased incidence rate of ~ 3% annually.


Drugs and
In addition, all studies were reported in accordance with the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines. Mahlavu cells prepared in DMEM (10,000,000 cells/mouse) were injected subcutaneously (SC) to the flank of 8-16 weeks old male nude mice as described previously 35 . Drug treatment was initiated once the tumor volume reached 150mm 3 . The subjects received compound 7b (100 mg/kg) suspended in simple syrup (16 g glucose in 9 g ddH 2 O) by oral feeding, and the control group of mice received only 100 µl simple syrup twice a week for 21 days. Nude mice were not treated for the following 3 weeks and imaged with Magnetic Resonance Imaging (acquired with 3-TESLA Siemens MAGNETOM Trio, UMRAM Center, Bilkent University) following intraperitoneal (ip) injection of anesthesia regimen consisting of 10 mg/kg xylaxin and 90 mg/kg ketamine. Statistical analysis. Data were obtained from three independent experiments, and all experiments were carried out with n ≥ 3 biological replicates. Statistical analysis for in vitro data was done using a Student's t-test. All in vivo experimental data were analyzed using ANOVA, n = 5-6 mice/group (Graphpad Prism version 7.0, or Microsoft Excel). * p < 0.05, ** p < 0.01, *** p < 0.001.

Cell growth inhibition, apoptosis, and cell cycle arrest induction by compound 7b. A label-
free real-time cell monitoring system (RT-CES) that can measure cell growth by electrical impedance detection was used to determine the real-time anticancer activity of compound 7b. It was demonstrated that compound 7b inhibits cell growth in a dose-and cell line-dependent manner, similar to our initial results with the SRB assay (Fig. 3a). Further experiments were performed to decipher the mechanism underlying this activity. In the presence of compound 7b, cleaved Poly-ADP-ribosyl-polymerase (PARP) fragments indicative of activated apoptotic pathway activation, were visible in most of the HCC cells after 24 h (Fig. 3b). The percentage of apoptotic cells increased significantly, as shown by Annexin-V/PI staining ( Supplementary Fig. S1a). Furthermore, caspase-9 and caspase-3 cleavage was also observed, indicating activation of intrinsic apoptotic pathway upon 7b treatment in HCC cells (Supplementary Fig. S1b). To further enlighten the mechanism beneath the apoptotic cell death in these cells, cell cycle distribution in the presence of compound 7b was examined by flow cytometry analysis of propidium iodide-stained cells. Compound 7b induced dose-dependent G2/M arrest in HCC cell lines after 24 h (Fig. 3c). In addition, 7b caused a significant increase in p53 and p21 expression levels and a minor decrease in the levels of CDK1 in HCC cells (Supplementary Fig. S2). www.nature.com/scientificreports/ stress-inducing dose 41 . Compound 7b did not induce ER-stress-specific XBP-1 splicing in these cells, unlike TN (Supplementary Figure S3). Then, we tested whether oxidative stress upon ROS accumulation was triggered by compound 7b in Huh7 and Mahlavu cells, which were treated with increasing concentrations of compound 7b (1.5 and 3 µM) for 8, 12, and 24 h. In the presence of compound 7b, the accumulation of ROS was detected significantly in both Huh7 and Mahlavu cells by DCFH-DA staining, which was in parallel quantified and statistically analyzed by flow cytometric analysis ( Fig. 4a and b). Additionally, ROS induction was significantly decreased once the cells were pretreated with NAC prior to 7b treatment (Fig. 4c). NAC pretreatment decreased   Fig. S4), which further supported the compound's mechanism of action as oxidative stress-induced apoptosis. It is well described in the literature that activation of c-Jun NH2-terminal kinases (JNKs), also known as stress-activated protein kinases (SAPKs), is a typical response to many forms of stress 42,43 . Therefore, we checked the levels of activated JNK and its downstream element c-Jun, a transcription factor phosphorylated by JNK1 and JNK2, in HCC cells treated with 7b, or JNKInhV relative to DMSO treated cells for 24 h. We observed a significant increase in the phosphorylated-JNK and phosphorylated-c-Jun protein levels upon compound 7b treatment (Fig. 5a). We tested the effect of 7b on the regulation of p38, another ROS-mediator-activated protein 44 , and found that phospho-p38 levels also increase significantly upon 7b treatment in HCC cell lines (Fig. 5a). To further investigate the effects of ROS accumulation on the upstream components of the JNK signaling pathway, levels of MKK-4, MKK7 45 and apoptosis signal-regulating kinase 1 (ASK1) proteins 46,47 were analyzed. We found that compound 7b increased the phospho-MKK7 protein levels (Fig. 5b) but did not change the phospho-MKK4 levels in HCC cells (Supplementary Fig. S5). Furthermore, 7b also altered the phosphorylation status of its upstream activator ASK1 (Fig. 5b). In contrast, NAC pretreatment reduced levels of JNK phosphorylation in HCC cells, further supporting our findings that JNK pathway activation is caused by ROS accumulation (Supplementary Fig. S6). To further analyze the role of JNK pathway activation and apoptosis induction by 7b, HCC cells were treated with JNK inhibitor together with 7b for 24 h and apoptotic cells were measured by Annexin-V/ PI staining. Cells were partially resistant to apoptosis in the presence of JNK inhibitor compared to cells treated with 7b alone (Supplementary Fig. S4).

Induction of ROS accumulation and activation of
In vivo anti-tumor activity of compound 7b in mouse xenografts. Mahlavu cell xenografts in nude mice were used to determine the in vivo anti-tumor effect of compound 7b. Mahlavu cells were selected due to their significant response to compound 7b and their poorly differentiated and highly metastatic nature 48 . We observed that compound 7b increased the overall survival of nude mice (Fig. 6a and Supplementary. Fig. S7). Moreover, the tumor volumes were significantly decreased in the compound 7b treated group, as demonstrated by the representative MRI images (Fig. 6b). Altogether, our in vivo data revealed that compound 7b is orally tolerable in nude mice and is a novel and potentially effective drug candidate against HCC. , are known to be capable of reconstituting the tumor by themselves, triggering metastatic events, and enhancing drug resistance in cancer cells 49 . Therefore, we tested compound 7b for its effect on enrichment LCSCs, which can be quantified by detecting CSC markers found on the surface of these cells. For this purpose, Huh7 and Mahlavu cells were treated with compound 7b and other inhibitors such as sorafenib, DAPT, or DMSO control at IC 50 concentrations for 72 h. Cells that remained viable after treatment were collected, and the expression of LCSC markers CD133 and EpCAM (for Huh7) and CD90 (for Mahlavu) were evaluated by flow cytometry. Results have revealed that compound 7b was able to decrease the CD133 + / www.nature.com/scientificreports/ EpCAM + population in Huh7 cells and the CD90 + population in Mahlavu cells significantly (Fig. 7a). Besides, sorafenib was known to enrich the LCSC population, as demonstrated previously 36 . To test the combinatory effect of compound 7b with sorafenib, Huh7 cells were treated with the increasing concentrations of both compounds simultaneously for 72 h. Flow cytometry analysis and the sphere formation assay have shown that compound 7b alone and, in combination with sorafenib, reduced the LCSC ratio and the sphere formation capacity of Huh7 cells. (Fig. 7b).

Effect of compound 7b on cell migration.
To identify the effect of compound 7b on the migration capacity of HCC cells, a real-time cell migration system (RT-CES, DP system, xCELLigence) was used. Huh7 and Mahlavu cells were treated with taxol as a positive control for cell migration inhibition or with compound 7b (Huh7: 2 µM, Mahlavu: 3 µM) for 24 h. Comparable with the effect of taxol, compound 7b inhibited the migration of both cell lines compared to the DMSO treated cells (Fig. 7c). Altogether, 7b could effectively interfere with the drug resistance-related cellular mechanisms such as stemness and migration.

Discussion
HCC is one of the most common and deadly cancers in the world. Sorafenib, which is a multi-kinase inhibitor approved by FDA was reported to prolong survival of advanced stage HCC patients by only 3 months 50 .
Although second-line treatment options such as Regorafenib were also studied for patients who are intolerant to sorafenib, drug resistance has become a critical problem for these patients due to the highly heterogeneous molecular nature of HCC.
Recent studies have reported that NSAIDs effectively decrease the incidence and mortality of various types of cancers 12,51,52 . In this study, we demonstrated that compound 7b, a synthetic 1,2,4-triazolo[3,4-b]-1,3,4-thiadiazine NSAID derivative, is a potent anticancer agent for HCC cells in vitro and in vivo, which was previously reported to have promising analgesic and anti-inflammatory activities 25,29 . Among the reported numerous biological effects, accumulation of reactive oxygen species (ROS) is considered as the underlying mechanism of the anticancer potential of NSAIDs 53,54 . Compatible with the literature, we have demonstrated that ROS accumulation induces growth inhibition, apoptosis, and G2/M cell cycle arrest in the presence of 7b (Figs. 3 and 4). We also examined signaling pathways activated by oxidative stress and found that activation of JNK and p38 as well as the downstream protein c-Jun was induced upon 7b treatment in HCC cells. (Fig. 5a). Further analysis of the upstream components of the JNK pathway revealed that compound 7b treatment causes cell line-specific activation of MKK7 protein (Fig. 5b) as well as dynamic regulation of phosphorylation sites on the ASK1 protein (Fig. 5b). Our in vivo data supported our findings on anticancer activity of 7b, where orally administered compound increased the overall disease-free survival for more than 4 months and reduced tumor size significantly in Mahlavu xenografts in nude mice (Fig. 6).
One of the most important factors associated with drug resistance in HCC is the presence of cancer stem cells. Conventional therapies fail to affect slowly dividing stem cell-like cancer cells and mainly target rapidly dividing tumor cells 55 . CSCs can reconstitute the tumor by themselves and manage to acquire metastatic features to migrate to distant organs 56,57 . Hence, we have also tested the compounds against LCSC and revealed that  www.nature.com/scientificreports/ compound 7b was also effective in inhibiting the enrichment of LCSCs (Fig. 7), meaning that compound 7b is not solely active on tumor cells (non-stem), but also tumor-initiating cells in HCC. We also tested the effect of compound 7b on the migration capacity of HCC cells, since CSC activity is associated with the migration and metastatic capacity of cancer cells 58 . 7b could effectively inhibit the migration of Huh7 and Mahlavu cells, comparable to the effect of Taxol, which is known as a microtubule stabilizing agent 59 that is widely used as a positive control for inhibition of migration in cancer cells 60,61 .
In the last decade, the anticancer and anti-metastatic effects of a well-defined NSAID, aspirin, has been described in cancer including HCC 62 . It is reported that by the induction of metabolic and oxidative stress in HepG2 cells, aspirin causes apoptosis through mitochondrial dysfunction 63 . In another study, aspirin was shown to attenuate pro-metastasis caused by sorafenib by upregulating the tumor suppressor HTATIP2 in nude mice xenografts 64 . Yet, the use of aspirin in treatment of cancer and its prevention remains uncertain due to the problems with the optimal dosage of aspirin and its serious side-effects such as gastrointestinal bleeding, increased uric acid, and coagulation inhibition 65,66 . Since these drugs are not designed to treat or prevent cancer progression, designing and developing novel NSAIDs that target cancer-related mechanisms is very crucial and can be considered as a new class of anticancer pharmaceutical agents such as non-steroid anti-inflammatory chemotherapeutic drugs (NSAICD).
In conclusion, our results revealed that 7b, a synthetic 1,2,4-triazolo[3,4-b]-1,3,4-thiadiazine NSAID derivative, is a promising compound with anticancer and anti-stem cell activities against HCC cells in vitro and in vivo. The anticancer effect was attributed to the induction of oxidative stress, cell cycle arrest, and eventually apoptosis through the JNK pathway regulation (Fig. 8). These findings highlight the potency of 7b as a new NSAICD, bearing a thiadiazine core, to be considered a promising anticancer drug for HCC patients and deserves further analysis. Figure 8. Illustration of molecular mechanisms involved in response to compound 7b treatment in HCC cell lines. 7b blocks cell proliferation through induction of ROS accumulation and JNK pathway activation which further stimulates transcription of genes related to apoptosis and cell cycle arrest. As a result, G2/M arrest (through the regulatory effect of p53 and p21), and apoptosis (through intrinsic pathway) are observed. 7b is also a potent compound acting on cancer stemness and migration capacity of HCC cell lines. This figure was created with BioRender.com. www.nature.com/scientificreports/ Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.