Furanocoumarin Notopterol: Inhibition of Hepatocellular Carcinogenesis through Suppression of Cancer Stemness Signaling and Induction of Oxidative Stress-Associated Cell Death

Background: Hepatocellular carcinoma (HCC) remains an aggressive malignancy with a poor prognosis and a leading cause of cancer-related mortality globally. Cumulative evidence suggests critical roles for endoplasmic reticulum (ER) stress and unfolded protein response (UPR) in chronic liver diseases. However, the role of ER stress in HCC pathogenesis, aggressiveness and therapy response remains unclear and understudied. Objectives: Against this background, the present study evaluated the therapeutic efficacy and feasibility of notopterol (NOT), a furanocoumarin and principal component of Notopterygium incisum, in the modulation of ER stress and cancer stemness, and the subsequent effect on liver oncogenicity. Methods: An array of biomolecular methods including Western blot, drug cytotoxicity, cell motility, immunofluorescence, colony and tumorsphere formation, flow-cytometric mitochondrial function, GSH/GSSG ratio, and tumor xenograft ex vivo assays were used in the study. Results: Herein, we demonstrated that NOT significantly suppresses the viability, migration, and invasion capacity of the human HCC HepJ5 and Mahlavu cell lines by disrupting ATF4 expression, inhibiting JAK2 activation, and downregulating the GPX1 and SOD1 expression in vitro. NOT also markedly suppressed the expression of vimentin (VIM), snail, b-catenin, and N-cadherin in the HCC cells, dose-dependently. Treatment with NOT significantly attenuated cancer stem cells (CSCs)-like phenotypes, namely colony and tumorsphere formation, with the concomitant downregulation of stemness markers OCT4, SOX2, CD133, and upregulated PARP-1 cleavage, dose-dependently. We also demonstrated that NOT anticancer activity was strongly associated with increased cellular reactive oxidative stress (ROS) but, conversely, reduced mitochondrial membrane potential and function in the HepJ5 and Mahlavu cells in vitro. Our tumor xenograft studies showed that compared with sorafenib, NOT elicited greater tumor growth suppression without adverse changes in mice body weights. Compared with the untreated control and sorafenib-treated mice, NOT-treated mice exhibited markedly greater apoptosis ex vivo, and this was associated with the co-suppression of stemness and drug-resistance markers OCT4, SOX2, ALDH1, and the upregulation of endoplasmic reticulum stress and oxidative stress factors PERK and CHOP. Conclusions: In summary, we demonstrated for the first time that NOT exhibits strong anticancer activity via the suppression of cancer stemness, enhanced endoplasmic reticulum stress and increased oxidative stress thus projecting NOT as a potentially effective therapeutic agent against HCC.


Introduction
Regardless of the remarkable advances made in hepatocellular carcinoma (HCC) diagnostics and treatment strategies, HCC still ranks as the sixth-most-common human malignancy and the third-commonest cause of cancer-related mortality, with over 905,000 new cases and more than 830,000 deaths, respectively, in 2020 alone [1,2]. An increased risk of HCC has been associated with dysregulated signaling pathways, coupled with concomitant oncogene activation/overexpression, and downregulated tumor suppressors in the liver cells; all of these are characteristic of chronic hepatitis B and C (HBV, HCV), addiction to alcohol, dietary toxins, and metabolic liver disease, such as nonalcoholic fatty liver disease (NAFLD) [3,4]. Dysregulated signaling has provided a bedrock for the emergence of targeted therapy as the standard of care for metastatic late-stage HCC in the last decade [5]. Since its approval by the Food and Drug Administration (FDA) over a decade ago, sorafenib has remained the mainstay of treating patients with HCC, until recently with the advent of new generation small molecule inhibitors of tyrosine kinases such as lenvatinib, regorafenib, ramucirumab, and cabozantinib, which have also demonstrated non-inferior therapeutic and/or prognostic effects [5,6]. Nonetheless, HCC remains a fatal malignancy with a high recurrence rate and is often characterized by chemoresistance [3][4][5][6].
The survival benefits of current therapeutic strategies for patients with HCC, including surgical resection and local ablation, are limited by about a 70% 5-year recurrence rate [7]. Unfortunately, resistance to therapy and cancer recurrence after initial treatment remains the greatest causes of HCC morbidity and mortality [7,8], and these cannot be dissociated from the activities of cancer stem cells (CSCs) [8]. CSCs are a small subpopulation of tumor cells with the intrinsic ability to self-renew, modulate cell differentiation, and enhance tumorigenesis [7][8][9]. Accruing evidence continues to highlight the important role of cancer stemness in the development and progression of HCC [8], and targeting these CSCs is increasingly seen as a potentially effective anticancer therapeutic strategy, including for HCC [8,9].
There is increasing evidence that some HCC environmental risk factors, namely HBV, HCV and alcohol addiction, promote liver carcinogenesis by enhancing oxidative stress [10]. Malignant cells predominantly use reactive oxygen species (ROS) and ROSassociated signaling ensuing from nutrient deprivation and hypoxia, which characterize the permissive tumor microenvironment essential for the development and progression of HCC [10]. Conversely, there are also reports of therapy-induced oxidative burst, or so-called ROS burst, resulting in apoptotic or autophagic cell death [10]. ROS is defined as a group of very reactive molecules that are known to regulate important signaling pathways [11]. ROS accumulation plays a critical role in signaling that drives cell cycle progression and cell proliferation. More so, increased ROS production or accumulation irreversibly alters target cellular macromolecules and results in intracellular damage associated with pathological states, including neurodegenerative diseases, cancer, and cell death [10][11][12]. However, as noted by Perillo et al., "ROS are also able to trigger programmed cell death (PCD)" [11]. The present study exploits the probable interplay between ROS production, oncogenic signaling, and cell death induction as a basis for the therapeutic activity and efficacy of notopterol. Notopterol (NOT) is a furanocoumarin and a principal bioactive component of Notopterygium incisum, a traditional Chinese medicinal herb with wide-spectrum pharmacological activity, including anti-inflammatory, pain-modulating, anti-rheumatism, and antihypertensive properties [13][14][15]. Intriguingly, a study has reported the anti-proliferative and pro-apoptotic effects of NOT in blood cancer [15], showing its capacity to interfere with the JAK-STAT or STAT3/NF-κB signaling pathway [13,16], which in turn leads to a decrease in the production of inflammatory cytokines and chemokines [13]. In certain HCC cases, the JAK-STAT signaling is abnormally activated, causing the dysregulation of downstream genes that may control processes such as cell survival, angiogenesis, stemness, immune surveillance, evasion, and metastasis [17]. As a result, exploring the potential of NOT as a therapeutic target for HCC presents a valuable research opportunity. However, it remains unclear if and to what extent NOT inhibits the development and/or progression of solid tumors, and more specifically, HCC. In this present study, the therapeutic effect of NOT was investigated, especially in terms of modulating HCC cancer stem cell (CSCs)-like phenotypes associated with aggressive tumor biology, as well as the mechanism underlying the CSCs-inhibitory and oxidative stress-inducing activities of NOT in HCC as a monotherapy and in comparison, with sorafenib, which is FDA-approved for advanced-stage HCC. From our current understanding and review of the existing literature, this study marks the initial examination of NOT's anticancer properties in hepatocellular carcinoma (HCC), along with its potential to impact cancer stem cells and oxidative stress signaling within HCC cells.

Cells and Cell Culture
Human liver cancer cell lines HepJ5 and Mahlavu generously provided by Dr. Chi-Tai Yeh (Department of Medical Research, Shuang Ho Hospital, New Taipei City, Taiwan) were cultured in Dulbecco's modified eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37 • C in a 5% humidified CO 2 incubator. Cells were sub-cultured at a cell confluence ≥ 95% or the medium was changed every 48 h.

Cell Viability and Drug Combination Assays
HepJ5 and Mahlavu cells were seeded in supplemented complete cell culture media at a density of 4 × 10 3 cells/well in triplicates in 96-well plates and incubated at 37 • C in 5% humidified CO 2 for 24 h before exposure to different concentrations of notopterol for up to 72 h. With the untreated wild-type cells serving as control, cell viability and proliferation were assessed by a sulforhodamine B (SRB) assay kit (ab235935; Abcam plc., Cambridge, UK) following the manufacturer's instructions. Optical density (OD) was measured at 495 nm wavelength in a SpectraMax microplate reader (Molecular Devices, Kim Forest Enterprises Co., Ltd., New Taipei City, Taiwan).

Transwell Matrigel Invasion Assay
Using a 24-well plate transwell system, we evaluated the invasion ability of the cells. The upper chambers of the transwell system were pre-coated with solubilized matrigel (BD Bioscience). After matrigel polymerization, 2 × 10 4 HepJ5 and Mahlavu cell lines were seeded into each upper chamber insert containing 200 µL FBS-free DMEM with different concentrations of notopterol, while the lower chamber contained 500 µL DMEM with 10% FBS serving as a chemoattractant. The medium was discarded after cell incubation for 24 h. The invaded cells on the lower side of the membrane were fixed with 10% formaldehyde for 20 min at room temperature and then stained with 0.2% (w/v) crystal violet solution for 20 min. The cells remaining on the upper side of the membrane were carefully removed with a cotton bud. The invaded cells were observed under a microscope and the total number of cells on the lower surface was counted.

Scratch Wound Migration Assay
Untreated control or treated Mahlavu and HepJ5 cells were seeded onto 6-well plates with complete growth media containing 10% FBS, and cultured to 100% confluence. The cell monolayers were scratched with a sterile yellow pipette tip to scratch the culture wells along the median axes, and fresh culture medium with or without notopterol was added. The wound closure migration images were captured at 0 and 48 h after the wound scratch, under a microscope with a 10× objective lens, and analyzed with the NIH ImageJ 149 software were downloaded from https://imagej.nih.gov/ij/download.html (accessed on 12 April 2023) and installed on a local computer.

Colony Formation Assay
Furthermore, 2 × 10 4 HepJ5 or Mahlavu cells were plated into 6-well cell culture plates and incubated at 37 • C for 2 weeks after treatment with or without notopterol. The HCC cells were then washed 3 times with cold 1× PBS, fixed with ice-cold methanol, stained with crystal violet dye, washed 3 times again with PBS, and dried at room temperature. The colonies formed were then evaluated and counted under a microscope. In each well, the total number of colonies (diameter ≥ 100 µm) was counted over 5 randomly selected fields in triplicate assays.

Hoechst33342 Side-Population (SP) Staining Flow Cytometry Assay
Hoechst33342 staining was used to evaluate the effect of notopterol on HCC side population cells. Cells were seeded at a concentration of 5 × 10 5 cells/mL and incubated for 48 hrs with the vehicle, notopterol, or sorafenib at indicated concentrations. Cells were harvested and washed thrice with ice-cold 1× PBS. Cells were then re-suspended in DMEM containing 2% FBS at 1 × 10 6 cells/mL containing Hoechst33342 (Cat.#B2261, Sigma-Aldrich, Merck KGaA, Darmstadt, Germany) at 5 µg/mL alone or combined with 50 µM inhibitor of ABC transporter, verapamil. After 90 min incubation at 37 • C, a BD LSRFortessa Cell Analyzer (BD Biosciences, San Jose, CA, USA) was used to perform flow cytometry analyses, using 488 nm excitation and fluorescence detection in log mode at the nominally far-red wavelengths > 695 nm. Hoechst33342 emission was initially split using a 610 nm dichroic short-pass filter, with red and blue emissions collected through 670/30 nm and 450/65 nm wavelength bandpass filters, respectively. Vehicle-treated and verapamil-treated cells served as the negative and positive control, respectively.

Detection of 2,7-Dichlorodihydrofluoroscin Diacetate (DCFDA) Intracellular Reactive Oxygen Species (ROS) Assay
Intracellular ROS level was measured using the ROS detection cell-based assay kit (DCFH-DA) (Cat.#601520, Cayman Chemical). The quantified principal component was cell-permeable DCFH-DA, which is easily oxidized to fluorescent dichlorofluorescein (DCF) by intracellular ROS. Briefly, HepJ5 or Mahlavu cells, seeded in 96-well plates and treated with indicated concentrations of notopterol for 24 h, were incubated with DCFH-DA at 37 • C for 20 min. The cells were then observed under a fluorescence microscope and measured at 488 nm and 525 nm excitation and emission wavelength, respectively, in a fluorescence microplate spectrophotometer (BioTek, SunPro International Inc., Taipei City, Taiwan).

Detection of Oxidative Stress Using MitoTracker ® Green Assay
Oxidative stress was detected using the MitoTracker ® Green FM (Cat.# M7514, Ther-moFisher Scientific Inc., Waltham, MA, USA) following the manufacturer's instructions. Briefly, HepJ5 and Mahlavu cells seeded and cultured in 96-well plates were treated with or without indicated concentrations of notopterol for 1 h and then stained with 20 nM MitoTracker ® Green for 30 min in a complete cell growth medium. Cells were then washed thrice with cold 1× PBS and imaged under the fluorescence microscope.

Detection of Intracellular Reduced Glutathione (GSH) Level
To measure the intracellular GSH level, the reduced glutathione (GSH) assay kit (colourimetric) (Cat.# MAK364, Sigma-Aldrich, Merck KGaA, Darmstadt, Germany) was utilized. The modified Tietze method, as described previously [18], was employed to determine the GSH levels in cells treated with or without the indicated treatment.

Tumor Xenograft Studies
Fifteen female BALB/c nude mice (6 weeks old, weighing 18-20 g) were purchased from BioLASCO (BioLASCO Taiwan Co., Ltd., Taipei, Taiwan). Animals were used according to protocols approved by the Laboratory Animal Committee of the Taipei Medical University (Protocol LAC-2021-0670). All mice were housed per experimental group in large cages (5 per cage) under specific pathogen-free (SPF) conditions: a temperature of 21 ± 7 • C, a humidity of 55 ± 5%, and a 12 h light/dark cycle with the lights coming on at 7.00 A.M.). The mice were fed a standard meat-free rat and mouse diet (SF00-100, Specialty Feeds, Western Australia, Australia), and freely accessed clean drinking water. The mice were randomly allocated into one of 3 experimental groups, namely control (n = 5), 30 µM Sora (n = 5), or 30 µM NOT (n = 5) for mice inoculated with cells treated with vehicle, 30 µM sorafenib or 30 µM notopterol, respectively, for 24 h before tumor inoculation. Tumor growth was monitored daily, and tumor size was measured every 72 h using callipers and the formula: (x × y 2 )/2, where x = longest diameter, and y = diameter perpendicular to x. Following 4 weeks, the mice were humanely euthanized, and their tumors were collected for analysis according to the standard protocol at the Laboratory Animal Center (LAC) of Taipei Medical University. A vaporizer was used to administer 5% isoflurane as the anaesthetic agent for euthanasia in small animals such as mice.

Data Analysis
Data are presented as means ± SEM (standard error of the mean) of experiments performed at least 3 times in triplicate. Statistical analyses were performed using GraphPad Prism ver. 7.0 for Windows (GraphPad Software, San Diego, CA, USA). Comparison between two groups was performed using a 2-sided Student's t-test, while the comparison of ≥3 groups was performed with the one-way analysis of variance (ANOVA). p-value < 0.05 was considered to be statistically significant.

Notopterol Suppresses Hepatocellular Carcinoma Cell Viability by Inhibiting JAK2 Activation and Enhancing Oxidative Stress
First, the impact of notopterol (NOT), which has a depicted chemical structure, molecular formula of C21H22O5, and molecular weight of 354.4 g/mol ( Figure 1A), was evaluated on HCC using the HepJ5 and Mahlavu cell lines. The cell cytotoxicity assays of NOT-treated cells compared with untreated control cells revealed that HepJ5 cells exhibited significantly reduced cell viability in a dose-and time-dependent manner. Specifically, exposure to 100 µM NOT for 24 h, 48 h, and 72 h resulted in a 33% (p < 0.001), 49% (p < 0.001), and 64% (p < 0.001) reduction in viability, respectively ( Figure 1B, left). Similarly, a 39% (p < 0.001), 60% (p < 0.001), and 79% (p < 0.001) reduction in Mahlavu cell viability was observed upon treatment with 100 µM NOT for 24 h, 48 h, and 72 h, respectively ( Figure 1B, right). Upon 48 h treatment with 30 µM NOT, in addition to the significant reduction in cell numbers, a transformation of the HCC cells from elongated spindle-like to cuboid morphology was observed ( Figure 1C). In parallel assays, treatment with 30 µM NOT was shown to markedly downregulate the expression levels of ATF4, p-JAK2, GPX1, CAT, and SOD1 proteins in the HepJ5 and Mahlavu cell lines in a time-dependent manner ( Figure 1D). These results suggest that notopterol suppresses HCC cell viability by inhibiting JAK2 activation and enhancing oxidative stress through the modulation of the aforementioned markers.

Notopterol Significantly Attenuates HCC Cell Migration and Invasive Capacity
As enhanced cell migration and invasion are key components of cancer metastasis and progression [19], the effect of NOT on these aspects was investigated. When compared with the untreated control, HepJ5 cells treated with 15 and 30 µM NOT exhibited a 53% (p < 0.05) and 62% (p < 0.01) decrease in migration, respectively (Figure 2A). A 47% (p < 0.01) and 68% (p < 0.01) reduction in migration was observed in Mahlavu cells treated with 15 and 30 µM NOT, respectively (Figure 2A). Similarly, treatment with 15-30 µM NOT significantly inhibited the invasion of HepJ5 (56-79%, p < 0.001) and Mahlavu (52-81%, p < 0.001) cells compared to the untreated control cells ( Figure 2B). Additionally, it was found that HepJ5 and Mahlavu cells treated with 15-30 µM NOT displayed a significant dose-dependent downregulation of VIM, Snail, β-catenin, and N-cadherin protein expression levels ( Figure 2C).

Discussion
HCC remains a fatal malignancy with a high recurrence rate and is often characterized by chemoresistance [3,6,22]. The characteristic HCC aggressive phenotypes, including chemoresistance, recurrence, and metastasis have been attributed to the presence of hepatocellular CSCs [7][8][9]22]. Hepatocellular CSCs essentially exhibit the capability to de-differentiate and self-renew and are implicated in the development and progression of HCC based on expressed specific cell surface markers, pluripotency biomarkers, and constitutively upregulated aldehyde dehydrogenase (ALDH) activity in HCC [23][24][25]. These hepatocellular CSCs often exhibit a quiescent phenotype with an intrinsic propensity for enhanced cell viability, self-renewal and cellular longevity by modulating oxidative stress markers, cancer stemness factors, and related biomarkers of cancer aggression, and thus the heightened interest in oxidative stress and CSCs as therapeutic targets in patients with metastatic HCC.
In the present study, it was demonstrated that notopterol suppresses hepatocellular carcinoma cell viability by inhibiting JAK2 activation and enhancing oxidative stress ( Figure 1). This is consistent with recent work by Wang Q et al. demonstrating that NOT ameliorates inflammation and rheumatoid arthritis by directly binding to the kinase domain of JAK2 and thereby inhibiting the Janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) signaling pathway [13]. Contextually, the aberrant activation of JAK2/STAT3 signaling characterizes various malignancies and is implicated in the tumorigenesis, angiogenesis, metastasis, and recurrence of many cancers, including HCC, and particularly those metastatic and/or recurrent cancers that are refractory to the standard chemotherapy [26,27]. It is clinically interesting that NOT not only inhibits JAK2 activation but that it enhances intracellular ROS-related oxidative stress. It is not impossible that by facilitating increased ROS flux, NOT elicits irreversible alteration of target macromolecules in the liver cancer cells, thereby inducing bio-cellular damage within the malignant hepatic cells, which consequently leads to a persistent redox homeostasis disequilibrium that culminates in HCC cell death. The glutathione redox cycle is a principal intracellular defence system consisting of GSH, which acts as a ROS scavenger and regulates the intracellular redox state, alongside glutathione peroxidase (GPx) and glutathione reductase (GR). The ability of any cell, and in this case, liver cancer cells, to regenerate GSH from its oxidized form GSSG is fundamental in buffering oxidative stress [10,11]; however, to the best of our current knowledge, this study demonstrated for the first time that NOT upregulated GSSG at the expense of GSH, thereby eliciting enhanced oxidative stress.
This study also showed that notopterol significantly attenuates HCC cell migration and invasive capacity, as well as effectively inhibits the cancer stem cells (CSCs)-like phenotypes of HCC cells (Figures 2 and 3). This is of clinical relevance, especially as the contemporary literature reports the strong association between hepatocellular CSCs, the development of HCC, and increased malignant traits of HCC such as enhanced invasiveness, resistance to treatment, early recurrence, easy metastasis, and poor prognosis [9,27,28]. The findings are consistent with the current knowledge that the plasticity of CSCs and the promotion of oncogenic activities such as migration and invasion, which are essential for HCC metastasis, more so as CSCs markers and the epithelial-to-mesenchymal transition (EMT), are closely associated with HCC metastasis [9,27,28]. It is postulated that by suppressing JAK2 phosphorylation, NOT inhibits JAK2/STAT3 signaling, enhances intracellular ROS burst, and destabilizes VIM, Snail, β-catenin, or N-cadherin oncogene interaction with the stemness marker SOX2 or OCT4, thus suppressing tumorsphere formation and oncogenicity and consequently leading to HCC cell death as expressed by upregulated cleaved PARP levels. This would be consistent with the demonstrated NOT-elicited inhibition of JAK2 activation, and the documented roles of JAK2/STAT3 signaling in oncogenicity, maintenance of cancer stemness, cellular survival under stress, and resistance to multiple anticancer therapies [8][9][10][11][24][25][26][27][28][29].
Furthermore, to the best of our knowledge, our study demonstrates for the first time that notopterol anticancer activity is mediated by disrupted stemness signals, increased intracellular ROS activity, and oxidative stress induction in HCC cells, in vitro and ex vivo (Figures 4 and 5). ROS acts as an important signaling molecule that tightly regulates CSCs plasticity and fate through the modulation of several intersecting intracellular signaling pathways [30]. Our findings corroborate the contemporary understanding that compared to normal cells, malignant cells may be more sensitive to ROS accumulation. Though rapid increases in intracellular ROS have been suggested to cause cellular transformation and tumorigenesis [29][30][31], in this study, it was demonstrated that NOT #stantially enhanced the ROS flux in HCC cells and that this substantial intracellular accumulation of ROS elicits irreversible cellular damage and consequent death. Interestingly, it was shown that the therapeutic potential of NOT was superior to that of sorafenib in terms of inhibiting side population or CSCs activity, inducing oxidative stress within malignant liver cells, and preventing tumor growth ex vivo ( Figure 5). Sorafenib, which is currently approved as a first-line treatment drug, was used as the positive control in this study. It functions by inhibiting the growth of tumor cells and the formation of blood vessels through targeting various serine/threonine and tyrosine kinases such as RAF1, BRAF, VEGFR 1, 2, 3, PDGFR, KIT, FLT3, FGFR1, and RET [32]. These are key players in several oncogenic signaling pathways. However, most patients eventually develop resistance to it. The ability of NOT to harness these biological features of ROS suggests the potential use of NOT as an effective ROS-mediated, CSCs-targeting, anticancer therapeutic agent for the treatment of patients with HCC, and that NOT can overcome therapy resistance.
In conclusion, as shown in the schematic of Figure 6, the findings presented herein are clinically relevant for HCC management because of the prevalent tendency of HCC cells to resist contemporary chemotherapeutic agents, as exemplified by a 0.25 response rate, no significant increase in patient overall survival [33], which is invariably associated with permissive TME, and the presence of hepatocellular CSCs, as demonstrated in our study. The present study, therefore, reports an effective anti-HCC therapeutic strategy that selectively kills cancerous liver cells, based on CSCs targeting and increased oxidative stress by exogenous ROS generation therapy. In this study, it was demonstrated that NOT effectively moderates the ROS environment in HCC cells both in vitro and ex vivo, and targets key CSCs markers via the modulation of oxidative stress. However, many key factors, such as the role of miRNAs in epigenetically controlling CSCs and ROS, as well as NOT's role in this process, have not been studied to uncover the molecular mechanism of NOT in overcoming the therapeutic challenge in HCC. Therefore, in future research, the aim will be to uncover the epigenetic role of key molecules in HCC, which may be modulated by the introduction of NOT, and to reveal the key molecular mechanisms of NOT in overcoming the therapeutic challenge.  Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/nu15112447/s1, Supplementary Table S1. The membranes were incubated in primary antibodies. Supplementary Table S2. List of gene-specific primers with detailed sequence used in this study. Supplementary Figure S1. Full Western-blotting result of the representative image in Figure 1D  Data Availability Statement: The datasets used and analyzed in the current study are publicly accessible as indicated in the manuscript.