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Tiliroside as a CAXII inhibitor suppresses liver cancer development and modulates E2Fs/Caspase-3 axis

Liver cancer is the fatal cause of cancer deaths worldwide due to its aggressiveness and lack of effective therapies. Tiliroside (C30H26O13) is an active compound extracted from herb plant Tribulus terrestris L., which has been used as alternative therapy in clinic practice. However, its therapeutic use against liver cancer has not been previously reported. Here, we showed that Tiliroside exerted significantly higher anti-proliferation effect on liver cancer cell lines Hep3B and SNU-449 than on liver normal cell THLE-3 cells or NC group, respectively, by using MTS assay. Results from colony formation, immigration and invasion assays support the anticancer efficacy of Tiliroside and its low-toxic property while treating liver normal cell THLE-3. 3D spheroid formation and CD133 expression level also displays its anti-stemness effect. It has been showed that Tiliroside may function as Carbonic anhydrases XII (CAXII) inhibitor and affects apoptotic E2F1/E2F3/Caspase-3 axis by using CAXII esterase activity assay, Human carbonic anhydrase 12 (CA-12) ELISA Kit, quantitative reverse transcription PCR (RT-qPCR) as well as CaspACE Assay System, respectively. In summary, we demonstrate for the first time that Tiliroside suppresses liver cancer development possibly by acting as a novel CAXII inhibitor, which warrant further investigation on its therapeutic implications.

Transfection assay and genetic silencing. Hep3B and SNU-449 cells were seeded at a density of 3 × 10 5 cells/6-cm dish and each incubated with the completed medium for 24 h in an incubator at 37 °C with 5% CO 2 . Then cells were transfected with a pCMV3-SP-N-HA vector (5 μg) containing the human CAXII (CA12) cDNA (Sino Biological, US) or the same amount of empty pCMVβ vector (control B) (VWR, US) by Lipofectamine 2000 (Thermo Fisher, US) according to the manufacturer's protocols. CAXII expression was confirmed after 24 h by RT-qPCR 49 .
For establishing CAXII knockout clones, Hep3B and SNU-449 were both transfected with 1 μg of CAXII CRISPR guide RNA vector plus 1 μg of vector coding for puromycin resistance by applying Metafectene Pro (Biontex, Munich, Germany), respectively, following the manufacturer's instructions(Bettina von Neubeck et al.2018). Cells were then selected by puromycin for 16 h. The stable knockout of CAXII was evaluated by RT-PCR.
MTS cell proliferation assay. Tiliroside was purchased from Sigma (USA) and prepared in DMSO (Sigma), stored in small aliquots at 20 °C. Cells were treated with either Tiliroside or DMSO (NC, negative control group) with different concentrations. Cell proliferation MTS assay was performed by adding 20 μL of MTS solution (PROMEGA, USA) into each well in a dark hood at different incubation time points (24 h, 48 h, 72 h, 96 h) following the manufacturer's instruction. Microplate Spectrophotometer (BIOTEK, USA) was used to detect the cells' absorbance at the wavelength of 450 nm. Triplicate were conducted for each condition at each time point. The proliferation inhibition rate was calculated based on the formula: inhibition rate = (1 − Absorbance of treated sample/Absorbance of control sample) × 100%.
To examine whether overexpression of CAXII can restore the inhibition of Tiliroside on cell proliferation, we transfected cells (Hep3B and SNU-449) with either CAXII or empty vector, followed by the treatment with 40 μM Tiliroside for 24, 48 and 72 h in 6 wells plates. The proliferation inhibition rates were calculated as mentioned above.
Colony formation assay. Nearly 2000 cells of either Hep3B or SNU-449 cells were paved in 6-well tissue culture plates for colony formation assays. After the cells were treated with either 40 μM Tiliroside (intervention group) or DMSO (NC, negative control group) for 24 h, the medium was replaced with a fresh complete medium. The plates were incubated in a humidified incubator at 37 °C with 5% CO 2 for 10 days. After each well was gently washed with DMSO, the cells were fixed by 4% paraformaldehyde (FD NEUROTECHNOLOGIES, USA) and dyed with crystal violet (SIGMA-ALDRICH, USA). The number of colonies with more than 20 cells was counted.
Wound healing assay. A wound healing assay was conducted in 6-well tissue culture plates with approximately 1 × 10 6 cells as seeds in each well. When the cell monolayer grew to 90% confluence, a wound scratch in each well was gently made by a 100-μL pipette tip. 40  The relative concentration was calculated by comparing the value of treatment and control groups. For CAXII esterase activity inhibition assay, all reagents were prepared as pervious study described 17 . Then, cell lysates were prepared for CAXII enzyme activity examination. The reaction was initiated by adding the substrate, and the enzyme activity was detected by monitoring the production of colored product, 4-nitrophenol (4-NP/pNP) (SIGMA-ALDRICH, USA), at 400 nm every 3 min for about 2 h at 37 °C. Absorbance of the spontaneous hydrolysis of the substrate alone and substrate in presence of Tiliroside, was subtracted from esterase activity in absence and presence of Tiliroside. Moreover, potent CAXII inhibitor (U-104) (EMD Millipore corporation, USA) (Cat No. 215901) in DMSO was added as a positive control following the manufacturer's instruction 24 .
The slope of the initial rate of enzyme activity was determined by plotting absorbance (Y-axis) and time (min) (X-axis), and then converted to the percentage of enzyme activity. Percentage of inhibition was calculated by setting the enzyme activity in absence of Tiliroside as 100%. The IC 50 values were obtained by non-linear leastsquares method using Prism 8 (version 8.3.0).

Western blot analysis.
Cell lysates were prepared and then loaded in a 10% polyacrylamide gel, followed by nitrocellulose membrane transference based on the standard Western blot assay protocol. 5% non-fat milk was added in the PBS for blocking was incubated in the mixture at 37 °C for 1 h. A corresponding antibody against a specific protein (anti-CAXII antibody was obtained from Abcam, USA,1/1000) was added for incubation at 37 °C for 2 h, and followed by the detection with an appropriate horseradish peroxidase conjugated secondary antibody (Ab205718, 1/20,000, ABCAM, USA) at 37 °C for 1 h. After the final PBS washing, signal was developed by ECL detection system and relative photographic density was quantitated by a gel documentation and analysis system (Alpha Imager 2000, ALPHA INNOTECH CORPORATION, USA).

RNA extraction and quantitative RT-PCR.
Total RNA was extracted from Hep3B, SNU-449 and THLE-3 cells using the RNeasy mini kit (QIAGEN, Germany) according to the manufacturer's instructions. The concentration and purity of total RNA were determined by using an Epoch microplate spectrophotometer (BIOTEK, USA). Total RNA was subsequently reverse-transcribed to cDNA templates using an AffinityScript multi temperature cDNA synthesis kit (Agilent technologies, CA, USA). The expression of E2F1, E2F3, CAXII and CD133 genes were determined using the SYBR Green PCR Kit (QIAGEN, Germany) on a 7500 Fast Realtime PCR System (LIFE TECHNOLOGIES, USA). The primers for CD133 were: forward, 5′-GAT TCA TAC TGG TGG CTG GGTGG-3′ and reverse, 5′-GCA GGT GAA GAG TGC CGT AAGT-3′ 25 , and carbonic anhydrase XII qPCR primer pairs(human) were obtain for Sino Biological (CN) (Cat: HP100073). Other primer sequences used in this study were described in our previous study 22 . Each sample was analyzed in triplicate, and the qPCR reaction conditions included one cycle of 95 °C for 15 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. The dissociation curve was run after the PCR amplification in each assay. The relative expression levels of a target gene mRNA between the treatment and control groups are expressed as a fold change relative to GAPDH using the 2 −ΔΔCt method.
Caspase-3 activity. The CaspACE Assay was conducted in a total volume of 100 μL in 96-well plates following the manufacturer's protocol. Cells of Hep3B, SNU-449 and THLE-3 cultured in 2D or 3D culture systems were treated with either 40 μM Tiliroside or the same amount of DMSO, respectively, as the induced apoptosis groups (48 h after intervention), and 3 μL Z-VAD-FMK inhibitor was added in intervention cells as the inhibited apoptosis groups. The mock groups were regarded as a normal control (NC). The plate was incubated at 37 °C in a humidified incubator with 5% CO 2 for 16 h. After the centrifugation of cell lysates, the cell supernatant fractions were harvested for CASP3 activity measurement 26 . In addition, the protein concentration of each sample www.nature.com/scientificreports/ was determined by the BCA protein assay (THERMO FISHER SCIENTIFIC, USA), and the pNA Calibration Curves were also made by a colorimetric assay system. CASP3 Specific Activity (SA) was calculated as described previously elsewhere 21 .
Statistical analysis. Numerical variables are shown as mean ± SD. One-way analysis of variance (ANOVA) was performed for group comparison with post-hoc Bonferroni test. The two-tailed unpaired Student's t tests were used for the difference comparison of two groups. Statistical significance was considered when P < 0.05 (two-sided). All statistics and figures were generated using GraphPad Prism 8.0 software (version 8.3.0).

Tiliroside suppresses proliferation, migration and invasion of HCC in vitro.
To explore the biological effect of Tiliroside on both liver cancer cell and liver normal cells in vitro, cancer cell lines of Hep3B and SNU-449, and normal cell line of THLE-3 were treated with either Tiliroside or DMSO, respectively. The inhibition rates of Hep3B in the presence of 40 μM Tiliroside ranged from 27.15 ± 3.7% to 52.27 ± 3.61% during 48 h to 96 h, and displayed significantly higher inhibition rates when compared to the 20 μM Tiliroside group with the range from 6.88 ± 0.8% to 11.57 ± 1.03% (P < 0.001, from 24 to 96 h) (Fig. 1A). The inhibition rate of SNU-449 in the Tiliroside group ranged from 35.39 ± 5.82 to 55.74 ± 1.85, also displayed significant differences to 20 μM Tiliroside group from 24 to 96 h (P < 0.001, from 24 to 96 h, respectively) (Fig. 1B). Moreover, for THLE-3, the inhibition rates of 40 μM Tiliroside group were significantly lower when compared to 80 μM group (Fig. 1C). The colony formation assays showed significantly reduced cell colony formation in Tiliroside groups with the relative efficiency of 61.15 ± 4.07% (P = 0.0002) in Hep3B and 75.51 ± 5.99% (P = 0.004) in SNU-449, respectively, compared with their NC groups (Fig. 1F,H).
The cell migration assay results displayed that the cells (Hep3B and SNU-449) in the Tiliroside group moved much slower than those in the NC groups ( Fig. 2A,B). As for Hep3B, at 24 h, the average width of wound gaps in the Tiliroside group was 94.37 ± 1.3% of its initial width compared to 45.72 ± 1.12% of the control group (P = 2.29 × 10 -6 ). At 48 h, the width in the Tiliroside group shrunk to 80.32 ± 2.13%, while its counterpart in   (Fig. 2C,D). Similarly, our cell invasion assays showed that the Tiliroside group displayed a weaker invasive ability than control group (Fig. 2E). In Hep3B cell, the average counts of invading cells in the control group was 208.33 ± 6.24 compared to 108.33 ± 5.14 of the Tiliroside (P = 8.84 × 10 -5 ). Additionally, the invading cells were 148 ± 5.1 in the control group compared to 92.67 ± 4.19 in the Tiliroside group (P = 0.0003) in SNU-449 cell (Fig. 2F).

Expression of CAXII effects the anti-tumor effect of Tiliroside on Hep3B and SNU-449. As
shown in RT-qPCR results, after transfection, in Hep3B cell, the relative expression levels of CAXII in the CAXII transfection group were 2.8 ± 0.07 folds vs. the non-transfection group (P < 0.001), and 0.924 ± 0.09 in the empty vector transfection group vs. non-transfection group (NS). Similarly, overexpression of CAXII was achieved by transfecting the cells with the CAXII vector in SNU-449 cell line (Fig. 1D). Moreover, in both Hep3B and SNU-449 cells, the relative expression level of CAXII in CAXII-KO (CAXII knock out) group was significantly deceased than that in none-KO (none knock out) and none transfection groups (P < 0.001), respectively (Fig. 1E).
Cells were treated with 40 μM Tiliroside after transfection. The proliferation assay showed that, at 24 h, in Hep3B cell, the inhibition rate (26.24 ± 4.15%) of CAXII-KO group was significantly lower than that of none transfection group (34.41 ± 4.01%) (P < 0.05) and none-KO group (34.9 ± 4.15%) (P < 0.05), respectively. Similar trends were observed at other time points. As for SNU-449, non-significant difference has been shown between CAXII-KO group and SNU-449 group (P = 0.054), after 24 h of Tiliroside treatment. However, the rest inhibition rates of CAXII-KO group were significantly decreased compared to counterparts in none transfection and none-KO groups (P < 0.05), respectively (Fig. 1G).
In Hep3B cells, inhibition rates of CAXII overexpression group were significantly higher than that in none transfection or empty vector transfection groups (P < 0.05), in all time points, respectively. However, as for SNU-449 cells, the inhibition rates of CAXII were found to be significantly raised compared to none transfection or empty vector transfection groups (P < 0.05), respectively, just at 48 and 72 h after Tiliroside treatment (Fig. 1G).

Inhibition of 3D spheroid formation and CD133 expression by Tiliroside.
To investigate the inhibition of 3D spheroid formation by Tiliroside, we cultured SNU-449 and Hep3B cells, both contain a luciferase reporter gene, using a hanging drop method. The dynamic changes of 3D spheroid formation are shown in Fig. 3A,B. Meanwhile, both cells treated by Tiliroside have been observed to be surrounded by ground-glass opacity. For Hep3B, a significant difference in the average 3D spheroid area between the Tiliroside group and the www.nature.com/scientificreports/ control group was shown from 48 to 120 h (Fig. 3C). For SNU-449 cell, the relative cell cross-section area of the Tiliroside group grew to 123.64 ± 11.63%, and showed significantly smaller compared to its counterparts of the control (153.64 ± 13.55%, P = 0.01) at 72 h, and this trend remained to the end point (120 h) which reached to 164.79 ± 18.72% in the Tiliroside group compared to 254.79 ± 27.64% of the NC (P = 0.02) (Fig. 3D). The luciferase reporter gene assays showed the similar results in 3D spheroid formation. The inhibition rates of Hep3B cells were steadily from 17.52 ± 2.74% to 56.4 ± 3.44% (24-96 h). Meanwhile, the inhibition rates of SNU-449 were from 11.75 ± 2.53% to 57.97 ± 12.19% (24-96 h) (Fig. 3E). Moreover, at 72 h, the rate of Hep3B (50.77 ± 2.26%) displayed significant difference with SNU-449 (41.03 ± 2.82%, P = 0.019) under the same amount of Tiliroside (Fig. 3E).

Tiliroside functions as a potential CAXII inhibitor. Molecular formula, weight and 3D structure of
Tiliroside are illustrated in Fig. 4A. CAXII was the top protein predicted target by the SwissTargetPrediciton tool with 100% probability.
As CAXII is a promising therapeutic target for cancer treatment, and impact of Tiliroside on the quantity of CAXII in the treated cells was tested using the Human carbonic anhydrase 12 (CA-12) ELISA Kit. Results showed that, in both 2D and 3D culture systems, Tiliroside has significantly decreased the expression of CAXII compared to NC group, in both Hep3B and SNU-449 at 24 h, 48 h and 72 h. For example, in 2D Hep3B experiment, at 48 h, the relative concentration of CAXII of Tiliroside group was significantly lower compared to that of NC group (0.23 ± 0.05, P < 0.001). Downregulation of CAXII has been also observed in SNU-449 and THLE-3 cells by Tiliroside intervention (Fig. 4B).
Moreover, CAXII esterase activity was determined after 1 h Tiliroside intervention. The activity curve was in a dose-dependent manner with an IC50 of 47.54 ± 3.6 μM and 34.67 ± 2.7 μM for Hep3B and SNU-449, respectively. For U-104, the activities were 6.3 ± 3.6 μM and 4.78 ± 3.6 μM for Hep3B and SNU-449, as shown in Fig. 4C,D. Downregulation of E2F1 and E2F3 by Tiliroside. RT-qPCR results showed that both liver cancer cell lines had higher E2F1 and E2F3 expression compared to normal liver cell line THLE-3 as reported previously (Fig. 4E) 27 . In Hep3B, the relative expression levels of E2F1 in the 2D and 3D Tiliroside groups were 0.34 ± 0.02, 0.44 ± 0.03 folds of the control groups, respectively (P < 0.001). The relative expression levels of E2F3 in 2D and 3D system were 0.436 ± 0.02 and 0.378 ± 0.01 folds of the control groups respectively (P < 0.001). The same trends were also detected in SNU-449 and THLE-3 cells (Fig. 4F,G). www.nature.com/scientificreports/ Figure 4. Tiliroside targeted CAXII enzyme as U-104 did and reduced its quantity and activity. Tiliroside also modulated the expression of E2F1, E2F3 whose relative expressions were higher in Hep3B and SNU-449 than in THLE-3. CAXII was predicted to be the potential targets of Tiliroside by Swiss target prediction tool (A). In both 2D and 3D culture systems, Tiliroside has significantly decreased the relative concentration of CAXII compared to NC group in both Hep3B, SNU-449 and THLE-3 at 24 h, 48 h and 72 h respectively (B). Dose-dependent inhibition of CAXII esterase activity by Tiliroside showed the IC50s were 47.54 ± 3.6 μM and 34.67 ± 2.7 μM for Hep3B and SNU-449, respectively (C), and that for U-104 were 6.3 ± 3.6 μM in Hep3B and 4.78 ± 3.6 μM in SNU-449 (D) (The concentration showed x axis stands for 0.5, 1.5, 5,10,20,80 μM, respectively). The relative expression levels of E2F1 and E2F3 were significantly higher in Hep3B and SNU-449 compared to that in THLE-3 (E). The relative expression levels of E2F1 (F) and E2F3 (G) in Hep3B, SNU-449 and THLE-3 cells were significantly downregulated by 40 μM Tiliroside in both 2D and 3D culture systems. Data are presented as the mean ± standard deviation (SD); NS not significant, NC negative control. www.nature.com/scientificreports/ Tiliroside promote caspase-3 activity. The CASP3 activities in the Tiliroside group was significantly higher compared to either the inhibited apoptosis or blank group in both Hep3B and SNU-449 cells cultured in 2D and 3D, respectively (P < 0.001) (Fig. 5A,B). The caspase-3 SA also showed a similar trend that the Tiliroside group held a higher SA value than the control group (P < 0.05) (Fig. 5C). Moreover, for 2D systems cultured cells, the SA in Hep3B Tiliroside group (0.117 ± 0.006) didn't show significant difference with that in SNU-449 (0.098 ± 0.0012, P = 0.09). Same trend has also been detected in cell cultured in 3D systems. Moreover, the SA in THLE-3 was 0.08 ± 0.01 (NC group) compared to 0.16 ± 0.01 (40 μM Tiliroside) (P < 0.001).

Discussion
Tiliroside is a compound found in several plants such as Tribulus terrestris and Agrimonia pilosa Ledeb 9 , and its anticancer efficacy has been examined in this study. Our results demonstrated for the first time that Tiliroside had an anticancer activity for liver cancer as a novel CAXII inhibitor, reduced stemness and differentiation, and could modulated apoptotic axis E2F1/E2F3-Caspase3. Moreover, the anti-cell proliferation activity of Tiliroside was less evident for normal liver cells while exerting stronger anti-cancer effect on cancer cells, suggesting that Tiliroside could be a promising agent for liver cancer treatment.
In vitro cell line experiments showed that Tiliroside significantly inhibited the proliferation, migration and invasion in both Hep3B and SNU-449 cells. Consistently, a significantly reduced 3D spheroid formation and reduced CD133 expression levels of both cells suggested that Tiliroside had an inhibitory effect on tumor stem cells or tumor-initiating cells. For details, the colony formation assay allows longer time (10 days) to evaluate cell survival in response to various treatment conditions. As expected, about 30% inhibition rate was observed in cell proliferation assay and approximately 40% inhibition rate was detected in colony formation assay while the same dose of Tiliroside was used in both assays. Moreover, Tiliroside significantly reduced (> 50%) migration and cell-cell interaction of liver cancer cells as observed in the wound healing and Transwell invasion assays. Results from 3D spheroid formation assay and CD133,E2F1 and E2F3 expression displayed its inhibitive effect of Tiliroside on HCC stemness, providing warranty for further in vivo investigation on Tiliroside against HCC.
Potential molecular mechanisms underlying the anticancer effect of Tiliroside were also investigated. The top protein predicted as a target by the SwissTargetPrediction tool was CAXII (CA12) with 100% probability, suggesting Tiliroside as a potential CAXII inhibitor. This prediction was confirmed by ELISA assay and CAXII activity assay. Results showed that, in both 2D and 3D culture systems, Tiliroside had significantly decreased the quantity of CAXII compared to NC group, in both Hep3B and SNU-449 cells at 24 h, 48 h and 72 h. Furthermore, CAXII esterase activity was also decreased by Tiliroside and showed a dose-dependent manner. CAXII is a membraneassociated zinc metalloenzyme, and is also a transmembrane protein which has been found to be associated with cancer progression by changing tumor microenvironment 14 . It has been considered to be the biomarker of favorable prognosis in lung, cervical and breast cancers 28,29 . However, high CAXII expression was negatively associated with prognosis in patients with colorectal cancer, oral squamous cell carcinoma, renal cancer and brain cancer 13,14,30 . Moreover, high CAXII expression has been found to be associated with poor outcomes for patients with HCC by analysis of clinical data (TCGA), Kaplan-Meier analysis and multivariate analysis 27 . For genetic depletion of CA IX and XII showed 85% reduction of tumor growth in colorectal cancer xenografts 31 . CA XII has also been displayed to enhance invasion and migration of breast cancer cells by regulating metalloprotease (MMP-2 and MMP-9) expression and of the p38 MAPK dependent pathway 32 . Studies also reported that tumor cells frequently overexpressed pH regulators such as CAIX and XII, and CAXII gene expression was also associated with tumor grading, which indicated their possible roles in tumor malignancy. In the tumor micro-environment, CAXII can create and transport bicarbonate ions into the cells through anion exchangers and Na + /HCO 3 − co-transporters which further produces favorable intracellular pH for cancer development and unfavorable extracellular acidosis to normal stromal cells. Therefore, intracellular pH modulation by inhibitors or knockdown of CAXII has been considered to suppress cancer development 17 . Blocking the enzymatic activity of CAXII has also been thought as a novel and safe way to treat cancer 17 .
Moreover, it has been found that CAXII was abundantly and homogeneously expressed in most tumor cells including HCC cells, but was largely undetectable in none-tumor tissues and normal liver tissues 14,30 . Evidence has also shown that CAXII expression was a risk factor for a poor prognosis in terms of DFS for HCC patients, which indicated CAXII could serve as a therapeutic target 33,34 . Previous studies have reported that inhibition of CAXII can promote apoptosis, therefore suppress cancer malignancy and proliferation via different pathways in breast cancer cells and T-cell lymphoma cells 10,35,36 , which is in line with the findings in our study. Therefore, Tiliroside could function as a novel CAXII inhibitor and an emerging approach for HCC treatment.
As we previously reported 22,23 , high expression of E2F1/3 are observed in HCC and are related to poor prognosis of cancer patients (including HCC patients). E2F1,3/Caspase-3 axis is recently reported to be involved in cancer cell apoptosis with anti-cancer effect. In this study, we demonstrated that Tiliroside significantly inhibited the expression of E2F1, E2F3, and increased CASP3 activity in both Hep3B and SNU-449 cell lines. Apoptosisrelated genes E2F1 and E2F3 are oncogenes, which are also associated with cancer stemness 37,38 . Overexpression of E2Fs is frequently observed in advanced cancer diseases and aggravates chemoresistance 39,40 . Our previous study have shown that the expressions of E2F1 and E2F3 have negative correlations with liver cancer patient survival, as well as that high expressions of E2F1 and E2F3 were found in HCC when compared to normal liver tissues 22 . On the other hand, the lack of CASP3 can cause the resistance of cells to microenvironmental stress and treatments, thereby promoting tumorigenesis 41,42 . Recent studies have found the suppression of CASP3 by E2F1 and E2F3, therefore reduced cell apoptosis in many types of cancer [43][44][45][46][47] . As a critical effector in cell apoptosis, CASP3 activation caused by the growth factor withdrawal or initiation of the Fas/Apo-1 receptor can consequently trigger the programmed cell death 48 . Inactivation or low expression level of CASP3 are often observed in many types of cancer, and the lack of CASP3 can also lead to the resistance of cells to microenvironmental www.nature.com/scientificreports/