s2fieldsofstudy: Biology Medicine

text:
Expression and Clinical Significance of High‐Mobility Group AT‐hook 2 (HMGA2) in Osteosarcoma

Objective Although high‐mobility group AT‐hook 2 (HMGA2) has been shown to have crucial roles in the pathogenesis and metastasis of various malignancies, its expression and significance in osteosarcoma remain unknown. Here we evaluate the expression, clinical prognostic value, and overall function of HMGA2 in osteosarcoma. Methods Sixty‐nine osteosarcoma patient specimens within a tissue microarray (TMA) were analyzed by immunohistochemistry for HMGA2 expression. Demographics and clinicopathological information including age, gender, tumor location, metastasis, recurrence, chemotherapy response, follow‐up time, and disease status were also collected. After validation of expression, we determined whether there was a correlation between HMGA2 expression and patient clinicopathology. HMGA2 expression was also evaluated in osteosarcoma cell lines and patient tissues by Western blot, we analyzed the expression of HMGA2 in the human osteosarcoma cell lines MG63, 143B, U2OS, Saos‐2, MNNG/HOS, and KHOS. HMGA2‐specific siRNA and clonogenic assays were then used to determine the effect of HMGA2 inhibition on osteosarcoma cell proliferation, growth, and chemosensitivity. Results HMGA2 expression was elevated in the osteosarcoma patient specimens and human osteosarcoma cell lines. HMGA2 was differentially expressed in human osteosarcoma cell lines. Specifically, a relatively high expression of HMGA2 was present in KHOS, MNNG/HOS, 143B and a relatively low expression was in MG63, U2OS as well as Saos‐2. HMGA2 expression is correlated with metastasis and shorter overall survival. High HMGA2 expression is an independent predictor of poor osteosarcoma prognosis. There was no significant correlation between HMGA2 expression and the age, gender, or tumor site of the patient. HMGA2 expression is predominantly within the nucleus. The expression of HMGA2 also directly correlated to neoadjuvant chemoresistance. There was a significant reduction of HMGA2 expression in the siRNA transfection group. After the use of siRNA, the proliferation of osteosarcoma cells is decreased and the chemosensitivity of osteosarcoma cells is significantly increased. Conclusion Our study supports HMGA2 as a potential prognostic biomarker and therapeutic target in osteosarcoma.

Introduction
O steosarcoma is the most common primary bone tumor and principally affects children and adolescents 1 . Current osteosarcoma treatment regimens rely on aggressive combinations of surgery and chemotherapy 2 . Treatment strategies with adjuvant chemotherapy have dramatically improved the prognosis for osteosarcoma patients, from a 5-year survival rate of 20% up to 55%-70% in cases of localized lesions 3 . However, the treatments and subsequent survival rates for osteosarcoma patients have plateaued over the past four decades and there are no predictive biomarkers of metastasis, chemotherapeutic response, or survival in osteosarcoma. Thus, there is a clear need for novel biomarkers and treatment strategies to improve osteosarcoma patient outcomes.
The high mobility group (HMG) proteins are present in only 3% of the histone content by weight, which are grouped into three families: HMGA, HMGB, and HMGN, previously known as HMG-11-2, HMG-141-17, and HMG-I (Y) 4 . High-mobility group AT-hook 2 (HMGA2) is a member of the High Mobility Group (HMG) protein family 5 . The HMGA2 protein is a non-histone architectural transcription factor encoded by its named gene on chromosome 12q15 6,7 . It contains three basic DNA-binding domains (AT-hooks) that enable binding to adenine-thymine (AT)-rich sequences of DNA within the minor groove 8 . This structural feature determines the binding preference of HMGA2 to secondary AT-rich regions DNA grooves, causing ordered structural changes that affect the bound conformation of DNA substrates and functional interactions between transcription factors, leading to changes in chromatin structure, DNA replication, and gene transcription 9 , thus regulating the transcription of several genes through chromatin remodeling. In this manner, HMGA2 has instrumental roles in cell differentiation and proliferation, DNA damage repair, apoptosis, and senescence. HMGA2 is a crucial regulator in stem cell maintenance and embryonic development, which can enhance the self-renewal potential of neural stem cells 10 and is highly expressed in human embryonic stem cells interacted with nucleosomes 11 . And while HMGA2 is uniquely expressed in early developmental stages before having trace detection in adult tissues, it is aberrantly re-activated and overexpressed in cancers [12][13][14] . HMGA2 is involved in the tumorigenesis and progression of different malignancies, included but not limited to lung cancer, breast cancer, acute myeloid leukemia, ovarian cancer, clear cell renal cell carcinoma, head and neck cancer, hepatocellular carcinoma, pancreatic cancer, and gastric cancer [15][16][17][18][19][20] . This overexpression is associated with poor clinical features such as metastasis as well as shorter survival. Despite its notoriety in other malignancies, HMGA2 expression, clinical significance, and potential as a therapeutic target remain unclear in osteosarcoma. The purposes of our study are: (i) to analyze the relationship between HMGA2 and the development of osteosarcoma; (ii) to provide a theoretical basis for the reversal of chemotherapy resistance of osteosarcoma; (iii) to elucidate HMGA2 as a potential therapeutic target of osteosarcoma. We therefore examined the expression of HMGA2 in osteosarcoma, its correlation with patient clinicopathology, and its roles in osteosarcoma cell proliferation and chemosensitivity.

Materials and Methods
Osteosarcoma Sample Collection and Tissue Microarrays (TMA) The osteosarcoma TMA was constructed from 69 individual osteosarcoma patient specimens within a formalin-fixed paraffin-embedded (FFPE) block as previously described 21 .

Immunohistochemistry (IHC) Staining
The expression of HMGA2 was evaluated by IHC staining according to manufacturer instructions (Cell Signaling Technology, MA, USA). In brief, the TMA slides were baked for 1 h at 60 C before xylene deparaffinization and subsequent rehydration through graded ethanol (100% and 95%). Endogenous peroxidase activity was quenched by 3% hydrogen peroxide after heated epitope retrieval. Subsequently, the slide was blocked for 1 h with normal goat serum, and incubated with polyclonal rabbit antibody to human HMGA2 (Cell Signaling Technology, 1:50 dilution, in 1% bovine serum albumin PBS) overnight in a humidified chamber set at 4 C. Each step was succeeded by three TBS rinses. Following this, SignalStain ® Boost Detection Reagent (Cell Signaling Technology) and SignalStain ® DAB (Cell Signaling Technology) were utilized to detect the bound antibody. Finally, hematoxylin QS (Vector Laboratories, CA, USA) was used to counterstain all the sections and obtain clearer images of the osteosarcoma cell nuclei before final long-term preservation using VectaMount AQ (Vector Laboratories) section mounting.

Analysis of Immunohistochemistry Staining
Two independent investigators blinded to patient clinical information and tumor characteristics assessed and scored the IHCstained slides. HMGA2 expression was subsequently scored and divided up into five groups according to the percentage of cells showing positive nuclear staining: 1+, <10% of positive cells; 2+, 10%-25% of positive cells; 3+, 26%-50% of positive cells; 4+, 51%-75% of positive cells; 5+, >75% of positive cells. The low HMGA2 expression subset included groups 1+ and 2+ while the high-HMGA2 expression subset included groups 3+, 4+, and 5+ (Fig. 1). Next, we analyzed the complete tumor necrosis data of the specimens and divided them into one of two groups based on their percentage of tumor tissue necrosis; good response: ≥ 90% necrosis; poor response: <90% necrosis. HMGA2 staining images were obtained by a Nikon Eclipse Ti-U fluorescence microscope (Diagnostic Instruments Inc., MI, USA) with a SPOT RT™ digital camera (Diagnostic Instruments Inc).

Human Osteosarcoma Cell Lines and Cell Culture
The human osteosarcoma cell lines MG63, 143B, U2OS, Saos-2, and MNNG/HOS were obtained from the American Type Culture Collection (VA, USA). The osteosarcoma cell line KHOS was kindly provided by Dr. Efstathios Gonos (Institute of Biological Research & Biotechnology, Athens, Greece). All osteosarcoma cell lines were cultured at 37 C in a humidified 5% CO 2 atmosphere in RPMI 1640 (GE Healthcare Life Sciences, Utah, USA) supplemented with 10% fetal bovine serum (Sigma-Aldrich, MO, USA) and 1% penicillin/streptomycin.

Protein Preparation and Western Blot
Protein lysates from the osteosarcoma cells and tissues were extracted using 1Â RIPA lysis buffer (Sigma-Aldrich) and protease inhibitor cocktail tablets (Roche Applied Science, IN, USA). The protein concentrations were then calculated by the DC™ protein assay reagents (Bio-Rad, CA, USA) and a spectrophotometer SPECTRA max 340PC (Molecular Devices, Inc., CA, USA). Western blot analyses were performed using similar methods as previously described 22 . In brief, equal amounts of proteins were separated by an SDS-PAGE gel (Thermo Fisher Scientific, CA, USA) and subsequently transferred to a nitrocellulose membrane (Bio-Rad). The membranes were then blocked in 5% nonfat milk for 1 h and incubated with the following specific primary antibodies at 4 C overnight: HMGA2 (Cell Signaling Technology, 1:1000 dilution), β-Actin (Sigma-Aldrich, 1:500 dilution) at 4 C overnight. After being washed three times for 5 min with TBST in room temperature, the membranes were further incubated with goat anti-rabbit IRDye 800CW (926-32,211, 1:5000 dilution) or goat anti-mouse IRDye 680LT secondary antibody (926-68,020, 1:15,000 dilution) (Li-COR Biosciences, NE, USA) for 2 h at room temperature. Following another wash with TBST three times for 5 min, the bands were scanned by the Odyssey Infrared Fluorescent Western Blots Imaging System from Li-COR Bioscience, and Odyssey software 3.0 was used to quantify the bands.

Immunofluorescence Assay
The expression of HMGA2 in the osteosarcoma cells was visualized via immunofluo-rescence assay. The 143B and KHOS cells were seeded at a density of 2 Â 10 4 cells/well in 24-well plates for 3 days and fixed with 4% paraformaldehyde for 15 min. The cells were then permeabilized with icecold methanol and blocked with 1% bovine serum albumin (BSA). An HMGA2 primary antibody (Cell Signaling Technology, 1:200 dilution) and β-Actin (Sigma-Aldrich, 1:500 dilution) were then incubated with the cells at 4 C overnight.
After being washed three times with PBS, the cells were incubated for an additional 1 h with Alexa Fluor 488 (Green) conjugated goat anti-rabbit antibody (Invitrogen, NY, USA) and Alexa Fluor 594 (red) goat anti-mouse antibody (Invitrogen). Nuclei were counterstained with 1 μg/mL Hoechst 33342 (Invitrogen). The cell images were scanned using a Nikon Eclipse Ti-U fluorescence microscope (Diagnostic Instruments Inc.) equipped with a SPOT RT™ digital camera.

Knockdown of HMGA2 by siRNA Transfection and MTT Assay
Knockdown of HMGA2 was performed with specific siRNA transfection in the osteosarcoma cells. The human nonspecific siRNA and HMGA2 siRNA (5 0 -GGAAGAACG CGGUGUGUAA-3 0 ) were purchased from Sigma-Aldrich. The nonspecific siRNA served as a negative control. 143B and KHOS cells were seeded at a density of 4 Â 10 4 cells/ well in 12-well plates or a density of 2 Â 10 3 cells/well in 96-well plates. Increasing concentrations of HMGA2 siRNA (0, 10, 30, 60 nM) or nonspecific siRNA (60 nM) were then transfected into the cells using Lipofectamine RNAiMax reagent (Invitrogen) according to manufacturer instructions. After 3 or 5 days, the transfected 143B and KHOS cells had their protein extracted for Western blot quantification or assessment of cellular proliferation by MTT assay. At the end of the 5-day transfection period, 20 μL of MTT (5 mg/mL, Sigma-Aldrich) was added to each well of the 96-well plates, and the cells were incubated for an additional 4 h at 37 C in a humidified 5% CO 2 atmosphere. Finally, the resulting formazan product was dissolved with 100 μL of acid isopropanol and the absorbance was read at a wavelength of 490 nm on the SpectraMax Microplate Spectrophotometer (Molecular Devices, LLC). Experiments were performed in triplicate. Cell viability curves were fitted using GraphPad PRISM 8 software (GraphPad Software, CA). Additionally, HMGA2 expression of the processed cells was evaluated by immunofluorescence assays.

Clonogenic Assays
The clonogenic assay was conducted to assess the effect of HMGA2 on cell viability and proliferation capabilities. The osteosarcoma cell lines 143B and KHOS were first seeded into 12-well plates at 100 cells/well and then treated with different concentrations of HMGA2 siRNA (0,10, 30, 60 nM). After 10 days of incubation at 37 C in a humidified 5% CO 2 atmosphere, the colonies were fixed with methanol for 10 min, washed three times with PBS, and subsequently stained with 10% Giemsa stain (Sigma-Aldrich) for 20 min. Finally, the colonies were washed with flowing water and allowed to dry. A digital camera (Olympus, Tokyo, Japan) was used to capture the pictures of the stained colonies.

MTT Cytotoxicity Assays
The MTT assay was performed to identify the effects of HMGA2 expression knockdown on cellular proliferation and chemosensitivity in the osteosarcoma cell lines 143B and KHOS. Briefly, 143B and KHOS cells transfected with HMGA2 siRNA were seeded into 96-well culture plates at a density of 3 Â 10 3 cells/well. After 24-h of transfection, the cells were then treated with or without different concentrations of doxorubicin or cisplatin for 5 days. Afterwards, 20 μL of MTT (5 mg/mL, Sigma-Aldrich) was added to each well, and the cells were incubated at 37 C in a humidified 5% CO 2 atmosphere for an additional 4 hours. Finally, the resulting formazan product was dissolved with 100 μL of acid isopropanol and the absorbance was read at a wavelength of 490 nm on the SpectraMax Microplate Spectrophotometer (Molecular Devices). Experiments were performed in triplicate. Cell viability and dose-response curves were fitted using GraphPad Prism 8 software (GraphPad Software, CA, USA).

Statistical Analysis
GraphPad Prism 8 software (GraphPad Software, CA, USA) and SPSS 26.0 software (IBM Corp., Armonk, New York) were used for statistical analysis. All the data are presented as mean AE SD. Independent two-tailed Student's t-tests were used to determine the statistical significance for independent data. One-way ANOVA tests were used for multiple comparisons. Survival analysis was evaluated by Kaplan-Meier plots and log-rank tests. The relationship between HMGA2 expression and osteosarcoma clinicopathological parameters was assessed by the χ 2 test. The prognostic factors associated with overall survival were evaluated through the Cox proportional hazards regression model in a stepwise manner. The factors deemed statistically significant (P < 0.05) in the univariate survival analysis were evaluated in the multivariate analysis. The correlation analysis was investigated by a Spearman's rank correlation.
To validate whether HMGA2 expression held clinical significance, we assessed its correlation with osteosarcoma patient pathology and outcomes. There was no significant correlation with HMGA2 expression and patient age (P = 0.379), gender (P = 0.387), or tumor site (P = 0.505) ( Table 1). However, there was a significant difference in HMGA2 tissue expression between patients with metastatic disease and those with primary localized tumor (P = 0.0165) ( Table 1, Fig. 2A). Based on patient follow-up data, expression of HMGA2 was markedly higher in the osteosarcoma tissues of patients who later developed metastatic disease compared to those who did not (P = 0.014) (Fig. 2B). We also found HMGA2 expression correlated with patient survival status, as patients who did not survive (dead) had significantly higher HMGA2 expression compared to those who survived at last available follow-up (alive) (P = 0.006) (Table 1, Fig. 2C). Importantly, Kaplan-Meier analysis showed high HMGA2 expression patients had shorter overall survival than those with low HMGA2 expression (P = 0.006) (Fig. 2D).
We further discovered that the expression of HMGA2 was inversely correlated to the percentage of tumor necrosis post neoadjuvant chemotherapy. Among the 69 patients, 52 received neoadjuvant chemotherapy before surgery. Nine patients (17.3%) showed ≥90% necrosis (good response) and 43 patients (82.7%) demonstrated <90% necrosis (poor response) after neoadjuvant chemotherapy (Table 1). Of note, patients with a poor response had significantly higher HMGA2 expression compared to those with a good response (P = 0.002) (Table 1, Fig. 2E). As predicted, Kaplan-Meier analysis demonstrated a significantly higher overall survival rate in patients with a good response compared to those with a poor response (P = 0.0013) (Fig. 2F). These results support elevated HMGA2 expression as a correlate for subtherapeutic necrosis following neoadjuvant therapy in osteosarcoma.
We also applied a Cox regression analysis to determine whether HMGA2 expression was an independent prognostic factor for osteosarcoma patients. In the univariate Cox regression analysis, a high expression of HMGA2 (P = 0.007) and presence of metastasis (P = 0.016) were associated with shorter overall survival in osteosarcoma patients, whereas other clinicopathological features showed no correlation (Table 2). Importantly, multivariate Cox regression confirmed that high expression of HMGA2 was an independent predictor of shorter overall survival in osteosarcoma patients (P = 0.006) ( Table 2). Our results indicate high HMGA2 expression is an independent predictor of poor osteosarcoma prognosis.

HMGA2 Expression in Osteosarcoma Cell Lines and Tissues
To determine the potential roles of HMGA2 in osteosarcoma, we analyzed the expression of HMGA2 in the human osteosarcoma cell lines MG63, 143B, U2OS, Saos-2, MNNG/ HOS, and KHOS. Western blot analysis showed HMGA2 was differentially expressed in these cell lines. Specifically, a relatively high expression of HMGA2 was present in KHOS, MNNG/HOS, 143B, and a relatively low expression was in MG63, U2OS, and Saos-2 ( Fig. 3A and B). To exclude the possibility of HMGA2 expression being an artifact induced by in vitro propagation, we evaluated HMGA2 expression in eight fresh osteosarcoma specimens. High expression of HMGA2 was found in four of the eight tissue specimens (50.0%) ( Fig. 3C and D). Furthermore, we performed immunofluorescent assays in the 143B and KHOS cell lines and determined the HMGA2 protein was mainly localized to the cell nucleus (Fig. 3E). This immunofluorescence data was consistent with our osteosarcoma TMA findings, which demonstrated that HMGA2 expression is predominantly within the nucleus as well.
The Role of HMGA2 siRNA in Osteosarcoma Cell Western blot and immunofluorescent assays were performed to evaluate the expression of HMGA2 in osteosarcoma cell lines following HMGA2-specific siRNA transfection. Western blot assays revealed a significant reduction of HMGA2 expression in the 60 nM siRNA transfection group compared to the cell only and 60 nM non-specific siRNA control groups in both 143B ( Fig. 4A and B) and KHOS ( Fig. 4C and D) cell lines. Immunofluorescent assays also confirmed dramatically reduced HMGA2 fluorescence in the 60 nM siRNA transfection group compared to the cell-only or 60 nM non-specific siRNA control groups in both 143B and KHOS cell lines (Fig. 5).

The Decrease of Osteosarcoma Cell Proliferation Caused by HMGA2 Knockdown
Effects of HMGA2 expression on osteosarcoma cell proliferation and growth were accessed by HMGA2-specific siRNA loss of function studies. Following transfection with increasing concentrations of HMGA2 siRNA, a dose-dependent decrease in cell viability was detected in both 143B and KHOS cells, a finding which was absent within the cell-only and nonspecific siRNA (60 nM) transfected controls ( Fig. 6A and B). We also used clonogenic assays to determine 143B and KHOS cell clonogenicity following HMGA2 knockdown with siRNA. Our results confirmed a reduction of clonogenicity after transfection with HMGA2 siRNA (60 nM) compared to the cell-only and non-specific siRNA (60 nM) transfected controls in both 143B and KHOS cells (Fig. 6C).
The Increase of Osteosarcoma Cell Chemosensitivity Caused by HMGA2 Knockdown Similar to other oncogenes, overexpression of HMGA2 may incur a survival advantage of human cancer cells. In order to evaluate the effect of HMGA2 expression on chemosensitivity in osteosarcoma cells, MTT assays were conducted to compare the cell viability of 143B and KHOS cells treated with increasing concentrations of doxorubicin or cisplatin following HMGA2 knockdown. We chose doxorubicin and cisplatin as they are the most often used chemotherapeutics for osteosarcoma treatment within the clinic 23,24 . We found increased chemosensitivity and lower IC50 values in both 143B and KHOS cells following knockdown of HMGA2 expression by siRNA transfection (Fig. 7). Taken together, we conclude that HMGA2 may play a critical role in the osteosarcoma chemoresistance seen within the clinic.

Discussion
A lthough the overexpression of HMGA2 has been shown to significantly correlate with metastasis, chemoresistance, and poor prognosis in various human cancers 20,25-27 , its expression and prognostic value within osteosarcoma remain unclear. Our study shows that HMGA2 was highly expressed in 71.0% of osteosarcoma patient tissue specimens. In addition, 39 of 48 (81.8%) of the osteosarcoma patients with metastasis showed overexpression of HMGA2, which was notably higher than the tissue specimens of patients with localized cancer. This finding is consistent with previous functional studies, where ectopic expression of HMGA2 induced epithelial-mesenchymal transition (EMT), a fundamental step in tumor cells becoming metastatic 28 .
Furthermore, our study revealed the overexpression of HMGA2 to correlate with shorter overall survival for osteosarcoma patients. The 10-year survival rate for patients with high expression of HMGA2 was a dismal 36.60%, significantly lower than the 72.45% seen in patients with low HMGA2 expression. Our results are consistent with previous studies which also found overexpression of HMGA2 to correlate with worse prognosis in cancers of the breast, lung, pancreas, ovaries, as well as gastric cancer, colorectal cancer, and oral cancer 15,[25][26][27][28][29][30] . These findings are the first to support HMGA2 as a potential biomarker predictive of metastasis and poor prognosis in osteosarcoma.
The response of osteosarcoma to neoadjuvant chemotherapy is the key factor of patient prognosis 31,32 . As determined by histological analysis, the tumor necrosis rate remains the gold standard for judging the neoadjuvant chemotherapy response 33 . Patients with a tumor necrosis rate ≥ 90% are defined as being good responders and have a much longer overall survival rate than those with a tumor necrosis rate <90% (poor responders) 34 . Poor responders tend to have greater rates of chemoresistance, metastasis, and recurrence. Accordingly, there has been considerable interest in identifying a biomarker which distinguishes between poor and good responders in osteosarcoma. In our study, HMGA2 expression was significantly higher in poor responders and correlated with shorter overall survival. As predicted, the overall survival of good responders with low HMGA2 expression was longer than in poor responders. These data suggest that HMGA2 is predictive of postneoadjuvant chemotherapy necrosis rates and overall patient prognosis in osteosarcoma.
HMGA2 knockdown with siRNA has reportedly reversed the mesenchymal phenotype and decreased proliferation of pancreatic cancer cells 35 . Transfection of HMGA2 siRNA has also shown to suppress proliferation and growth of ovarian cancer cells overexpressing HMGA2, and decrease tumor xenografts in athymic nude mice treated with a HMGA2-targeting construct 36 . Similar effects have been observed in colorectal cancer and myeloid leukemia cells as well 37,38 . Our study also revealed that HMGA2 is expressed in osteosarcoma cell lines and fresh osteosarcoma tissues, with the cell lines showing a significant reduction of growth and viability following HMGA2 knockdown. The clonogenic assays also showed a reduction in size and number of osteosarcoma colonies following the silencing of HMGA2, further suggesting the importance of HMGA2 in osteosarcoma growth and division. These promising anticancer effects warrant future in vivo models for HMGA2 targeting.
Previous studies have shown that high expression of HMGA2 is associated with chemoresistance in several tumors [39][40][41] . Functionally, elevated levels of HMGA2 protects cancer cells against DNA breaks from the topoisomerase inhibitor irinotecan 42 . Overexpression of HMGA2 also enhances 5-fluorouracil resistance in colorectal cancer both in vitro and in vivo 39 , and attenuates sensitivity to PARP inhibitors in breast cancer 43 . Through downstream regulation of ERK1/2 signaling, HMGA2 also increases gemcitabine resistance 44 . In our study, knockdown of HMGA2 increased osteosarcoma cell sensitivity to doxorubicin and cisplatin. While further mechanistic study is needed, our data suggest that targeting HMGA2 in combination with commonly used osteosarcoma chemotherapeutics is a promising new treatment approach. Still, there are limitations in this article. A potential limitation of our study is lack of animal experiment. Another limitation of this study is lack of indepth research on the mechanism.

Conclusion
In summary, our study demonstrates that HMGA2 is overexpressed in the majority of osteosarcoma tissues and is associated with a worse clinical prognosis and chemoresistance for osteosarcoma patients. Inhibition of HMGA2 decreased osteosarcoma cell growth and proliferation while enhancing chemosensitivity. Our findings suggest that HMGA2 is a novel prognostic biomarker and a promising therapeutic target in the treatment of osteosarcoma, especially when used in combination with standard chemotherapeutics.


text:
Effect of taxifolin on cyclophosphamide-induced oxidative and inflammatory bladder injury in rats

The role of oxidative stress and inflammation in the pathogenesis of cyclophosphamide-related side effects has been demonstrated in previous studies. This study aimed to investigate the effect of taxifolin, due to its antioxidant and anti-inflammatory properties, on cyclophosphamide-induced oxidative and inflammatory bladder injury in albino Wistar rats. The taxifolin+cyclophosphamide (TCYC) group was given 50 mg/kg of taxifolin orally by gavage. Normal saline was used as a solvent for the cyclophosphamide (CYC) group and the healthy control (HC) group. One hour after taxifolin administration, 75 mg/kg of cyclophosphamide was intraperitoneally injected in the TCYC and CYC groups. This procedure was repeated once a day for 30 days. At the end of this period, biochemical markers were studied in the excised bladder tissues and histopathological evaluations were conducted. In the histopathological evaluation of the CYC group, severe epithelial irregularity, dilatation, congestion, and polymorphonuclear leukocyte accumulation in the vascular structures were observed. Additionally, the malondialdehyde (MDA), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) levels, the total oxidant status (TOS), and the oxidative stress index (OSI) values were significantly higher, and the total glutathione (tGSH) levels and total antioxidant status (TAS) were significantly lower in the CYC group in comparison to the HC group (P<0.001). Taxifolin reduced the cyclophosphamide-induced increases in the MDA, TNF-α, IL-1β, and IL-6 levels and the TOS and OSI values; it decreased the tGSH and TAS levels and reduced histopathological damage (P<0.001). Taxifolin may be useful in the treatment of cyclophosphamide-induced bladder damage.

Introduction
cyclophosphamide is a nitrogen mustard derivative anticancer drug that is mainly used in the treatment of malignancies, such as Hodgkin lymphoma and Non-Hodgkin lymphoma, lymphocytic lymphoma, small lymphocytic lymphoma, Burkitt lymphoma, and multiple myeloma [1]. breast cancer, diffuse neuroblastomas, retinoblastoma, and ovarian adenocarcinomas are also among the additional Fda-approved indications for its use [1]. As an effective immunosuppressive agent, cy-clophosphamide is prescribed for the treatment of autoimmune diseases, such as multiple sclerosis, as well as before transplantation to prevent transplant rejection and graft-vs-host complications [2,3]. However, nausea, bladder cancer, bone marrow suppression, increased opportunistic infections, and hematological and other organ toxicities have been observed during the use of cyclophosphamide [4]. urotoxicity-like hemorrhagic cystitis has also been reported [1,5]. one of cyclophosphamide's main metabolites, acrolein, is thought to be responsible for the drug's bladder toxicity, and Mesna is commonly | Exp. Anim. 2022; 71(4): 460-467 used to counteract it [6,7]. ıt is well known that, in some patients, Mesna does not properly protect the bladder from the harmful effects of cyclophosphamide. The increase in the level of malondialdehyde (mda), a result of lipid peroxidation (lPo), and the decrease in the level of the endogenous antioxidant glutathione (GSH) have been demonstrated to cause the toxic effects of cyclophosphamide on the bladder. It has also been suggested that cyclophosphamide-associated cystitis is associated with the expression of proinflammatory cytokines, such as nuclear factor-κb (NF-κb), tumor necrosis factor-alpha (TNF-α), interleukin 1 beta (IL-1β), and ıl-6 [1,8]. according to evidence reported in the literature, antioxidant and anti-inflammatory medications may be beneficial in the treatment of cyclophosphamideinduced bladder injury.
Taxifolin (3,5,7,3′,4′-pentahydroxy-flavanone) whose effects against cyclophosphamide-induced oxidative and inflammatory bladder injury are investigated in our study, is an antioxidant flavonoid [9]. taxifolin is found in the content of plants, such as milk thistle, onions, French maritime pine, and tamarind [10]. It has been proven that taxifolin has anti-inflammatory, antitumor, antimicrobial, and antioxidant properties [11]. It has been reported that the antioxidant effect of taxifolin is due to its inhibition of reactive oxygen species (ROS) production [12].
according to our extensive literature search, no previous studies have investigated the preventive effects of taxifolin against cyclophosphamide-induced oxidative and inflammatory bladder injury. Therefore, our study aimed to biochemically and histopathologically investigate the effect of taxifolin on cyclophosphamide-induced oxidative and inflammatory bladder injury in rats.

Materials and Methods
Animals the animals we used in the study were supplied from Erzincan binali Yıldırım University Medical Experiments application and Research center. a total of 18 male, albino Wistar rats weighing between 250 and 265 grams were used in the experiment. the animals were obtained from the Erzincan binali Yıldırım University medical experimental application and Research center. The animals were housed and fed in a suitable laboratory environment at normal room temperature (22°c) under appropriate conditions (12 h of light and 12 h of darkness). animal experiments were performed in accordance with the National Guidelines for the use and Care of Laboratory Animals and were approved by the local animal ethics committee of erzincan Binali Yıldırım University, Erzincan, Turkey (Ethics Committee No.: 2021/05, dated dec 30, 2021).

Chemicals
the cyclophosphamide (a vial containing 1 g of powder solution for infusion) used in the experiments was supplied from EİP Eczacıbaşı (İstanbul, Turkey), thiopental sodium was supplied from I.E. Ulagay (İstanbul, Turkey) and taxifolin was obtained from Evalar (Moscow, Russia).

Experimental groups
the rats used in the experiment were divided into three groups: a cyclophosphamide (cyc) group, a taxifolin + cyclophosphamide (tcyc) group, and a healthy control (Hc) group.

Experiment procedure
to implement this experiment, the tcyc group (n=6) was given 50 mg/kg of taxifolin orally by gavage. This dose of taxifolin has been used in animals before and has been found to be effective against oxidative liver damage [13]. Normal saline (0.9% Nacl) was used as the solvent for the cyc (n=6) and Hc (n=6) groups. one hour after taxifolin and solvent administration, 75 mg/ kg of cyclophosphamide was injected intraperitoneally in the animals in the tcyc and cyc groups. this procedure was repeated once a day for 30 days. at the end of this period, the animals in all three groups were killed with high-dose anesthesia (thiopental sodium 50 mg/kg) and the bladder tissues were removed. The MDA, total glutathione (tGSH), TNF-α, IL-1β, and IL-6 levels and the total oxidant status (toS) and total antioxidant status (TAS) were determined in the removed bladder tissues. The bladder tissues were also examined histopathologically. The biochemical and histopathological examination results were compared between the three study groups and evaluated.

Tissue MDA and tGSH determination
after measuring the weights of the tissues, samples were homogenized immediately for use for mda measurements. MDA measurements are based on the method used by Ohkawa et al., which includes the spectrophotometric measurement of the absorbance of the pink-colored complex formed by thiobarbituric acid and mda [14]. briefly, 25 µl of tissue homogenate were mixed with 25 µl of 80 g/l sodium dodecyl sulfate and 1 ml of combination solution (20 g/l acetic acid + 1.06 g 2-thiobarbiturate + 180 ml distilled water). The mixture was incubated for 1 h at 95°C. The mixture was centrifuged for 10 min at 4,000 rpm after cooling. the supernatant's absorbance was measured at 532 nm. The standard curve was obtained by using 1,1,3,3-tetramethoxypropane. tGSH measurement was made according to the method described [15].

Tissue TOS and TAS determination
the toS and taS results of tissue homogenates were determined using a novel automated measurement method and commercially available kits (Rel Assay Diagnostics, Gaziantep Turkey), both developed by Erel [16,17]. The measurement of different oxidants and antioxidant molecules separately is not practical. therefore, measuring the total oxidant or antioxidant capacities of a biological sample and relating them in a ratio, called index is preferred. one of the indexes used for this purpose is the "oxidative stress index (oSı)". the oSı value calculated according to the "OSI (Arbitrary Units) = TOS (nmol H2o2 eq/mg protein) / taS (nmol trolox eq/mg protein) × 100" formula.
TNF-α, IL-1β, and IL-6 analysis the weight of the tissue samples was measured; then the samples were rapidly frozen with liquid nitrogen and homogenized using a pestle and mortar. the samples were maintained at 2-8°C after melting. We added phosphate-buffered saline (pH 7.4), 1/10 (w/v). Then, the samples were vortexed for 10 s, centrifuged for 20 min at 10,000 × g and the supernatants were carefully collected. The levels of TNF-α (ng/l), IL-1β (pg/l), and ıl-6 (ng/l) were measured using a commercial elıSa kit supplied by Eastbiopharm Co., Ltd. (Hangzhou, china).

Histopathological analyses
The tissue samples were fixed with 10% formaldehyde for 72 h. Following the fixation process, the tissue samples were washed under tap water in cassettes for 24 h. the samples were then treated with conventional grade alcohol (70%, 80%, 90%, and 100%) to remove the water within the tissues. the tissues were then passed through xylol and embedded in paraffin. Four-to-five micron sections were cut from the paraffin blocks and hematoxylin-eosin staining was administered. the sections were photographed and assessed using the dP2-SAL firmware program and a light microscope (Olympus ınc., tokyo, Japan). From the serial sections taken, one central and five peripheral areas were selected for semiquantitative scoring, and degeneration criteria were scored in the selected areas for each subject. Urinary bladder tissue damage was defined as the presence of epithelial necrosis, dilatation/congestion, polymorphonuclear leukocyte (PMNL) infiltration, and edema. Each sample was scored for each criterion as follows: 0: indicated no damage; 1: mild damage; 2: moderate damage; 3: severe damage. Histopathological assessment and scoring were performed by a pathologist who was blinded to the experimental groups.

Statistical analysis
IbM SPSS Statistics for Windows, Version 22.0, released 2013 (ıBm corp., armonk, Ny, uSa) was used for the statistical analysis. the results are presented as mean ± SD. The significance of differences between the groups was determined using the one-way analysis of variance (ANOVA) test followed by Tukey's analysis. The statistical level of significance for all tests was considered to be 0.05.

Biochemical findings
As can be seen from Table 1, the amount of mda in the bladder tissue of the rats in the CYC group was significantly increased in comparison to the HC and TCYC groups, while the amount of tGSH decreased (P<0.01). There was no statistically significant difference between the Hc and tcyc groups in terms of the mda and tGSH levels (Table 1).

TOS, TAS, and oxidative stress index (OSI) analysis results
ın the cyc group, cyclophosphamide induced an increase in the TOS value of the bladder tissue and it decreased the taS value. the toS level was lower in the Hc and tcyc groups in comparison to the cyc group (P<0.001), and the taS value was higher (P<0.001, Table 1). Furthermore, the difference in the TOS values between the HC and TCYC groups was determined to be insignificant, despite the fact that there was a significant difference in TAS (P=0.046, Table 1). The OSI value was found to be significantly higher in the CYC group than the Hc and tcyc groups. However, the difference in the OSI value between the HC and TCYC groups was insignificant (P>0.05).

TNF-α, IL-1β, and IL-6 analysis results
TNF-α, IL-1β, and IL-6 levels in the bladder tissue of the animals in the CYC group increased significantly in comparison to the Hc and tcyc groups (P<0.001, Table 1). Taxifolin significantly inhibited the increase in the TNF-α, IL-1β, and IL-6 levels induced by cyclophosphamide. The difference in the IL-1β levels was found to be insignificant, although there was a significant difference in the TNF-α and IL-6 levels between the TCYC and Hc groups (P=0.002 and P=0.015 respectively, Table 1).

Histopathological findings
In the bladder tissues of the animals in the HC group, the stratified transitional bladder epithelium, connective tissue, muscle tissue underneath the epithelium, and the distribution and architecture of the blood vessels in the connective tissue and muscle tissue were all normal (Fig.  1). In the bladder tissues of the animals in the CYC group, in low magnification, epithelial dysregulation, grade-3 dilatation and congestion in the blood vessels in the underlying connective and muscle tissue, grade-3 polymorphonuclear leukocytes (PNl) accumulation in the blood vessels, and grade-2 PNL cell infiltration around the blood vessels were observed (Fig. 2). Furthermore, when the high magnification images of the CYC group were examined, the stratified transitional epithelium lost its continuity in some areas, and grade-3 necrotic cells appeared in the areas where it was continuous. ın the connective tissue underneath the epithelium, grade-3 edema and fibril separations were identified (Fig. 3).
conversely, in the samples from the tcyc group, the epithelium is continuous, the underlying connective tissue is free of edema, and the fibrils are in a normal arrangement. mild-to-moderate (grade-1 and grade-2) dilatation and congestion in the blood vessels, as well as grade-1 polymorphonuclear cell infiltration (PMNL), were observed in the connective tissue (Fig. 4). Table 2 presents the statistical analysis of histopathological findings in the Hc, cyc, and tcyc groups.

Discussion
In this study, taxifolin's effects on the oxidative and inflammatory bladder damage caused by cyclophosphamide in rats were studied biochemically and histopathologically. The balance between the oxidant and antioxidant systems in cyclophosphamide-induced oxidative  bladder injury has been studied previously [8]. the level of mda was increased and the level of tGSH was decreased in the animals with cyclophosphamide-induced bladder tissue injury, according to Abraham et al. [18]. as previously stated, RoS initiates the lPo reaction in the cell membrane, which results in the creation of toxic products, such as mda from lipids [19]. these findings suggest that cyclophosphamide may have increased the production of ROS in the bladder tissue. In a study supporting this, it was reported that cyclophosphamide increased the production of ROS in the bladder tissue [20]. The fact that the MDA levels in the bladder tissue were higher in the cyc group than the Hc group indicates that our experimental results are consistent with the data reported in the literature [21]. ıt is known that cyclophosphamide, which causes oxidative stress in bladder tissue, reduces endogenous antioxidant levels [22]. the decrease in the amount of tGSH in the bladder tissue to which we applied cyclophosphamide shows that our experimental results are in agreement with previous studies. tasdemir et al. reported that cyclophosphamide decreased the amount of tGSH and increases the oxidant parameters in the blad-der tissue [23]. ın the literature, an increase in the oxidant parameters and a decrease in the antioxidant parameters have been associated with ROS overproduction [24]. Superoxide, singlet oxygen, hydroxyl radical, and hydrogen peroxides are known types of RoS [25]. tGSH is an endogenous antioxidant with ROS scavenging ability. Disruption of the ROS/GSH balance results in the oxidation of biomacromolecules; this leads to cell cycle arrest and even cell death [26].
to evaluate total enzymatic and non-enzymatic oxidant and antioxidant parameters, toS and taS in the bladder tissue were analyzed and the OSI values were calculated. The TOS and TAS analysis results obtained from the cyc group and the Hc group verify that the level of MDA was increased in the bladder tissue and the level of tGSH was decreased. as is known, toS and TAS reflect the total effects of all antioxidants and oxidants in tissues [16,17]. our results and this information obtained from the literature reveal that the oxidant/antioxidant balance in the bladder tissue of the animals in the CYC group is maintained by the predominance of oxidants.
In our study, it was found that the levels of proinflam-   matory cytokines, such as TNF-α, IL-1β, and IL-6, significantly increased in the bladder tissue of rats in the cyc group in comparison to the Hc group. ın recent studies, it has been reported that the levels of TNF-α, IL-1β, and IL-6 in the bladder tissues of animals with hemorrhagic cystitis due to cyclophosphamide are increased in comparison to the healthy control group; it has also been confirmed histopathologically that these proinflammatory cytokines cause inflammation in the bladder tissue [27]. A recent study by Wróbel et al. revealed that TNF-α, IL-1β, and IL-6 are important factors in the pathogenesis of cyclophosphamide-associated cystitis [28]. There also appears to be a correlation between the increase in proinflammatory cytokines and the oxidant and antioxidant parameters in cyclophosphamide-induced bladder damage [27]. In our study, it was observed that significant histopathological damage developed in the bladder tissue of the CYC group, in which the oxidant and proinflammatory parameters increased and the antioxidants decreased. the animals treated with cyclophosphamide experienced severe dilation and congestion of the bladder blood vessels. It has been reported that cyclophosphamide causes congestion in animal bladder tissue [29]. However, no previous studies reported that cyclophosphamide caused vasodilation in animal bladder tissue, based on a histopathological examination. ın a patient who was treated with cyclophosphamide, it was found that capillary dilatation developed in the bladder mucosa in the cystoscopy performed [30]. many studies have supported our findings showing PMNL accumulation in the bladder tissue of the cyclophosphamide group. Abd-Allah et al. and moraes et al. reported that cyclophosphamide causes intense PMNL infiltration in rat bladder tissue [29,31]. as it is known, the accumulation of PmNl in tissues is an indicator of inflammatory reaction. In our study, edema, which is one of the main signs of inflammation, developed in the connective tissue of the bladder in the CYC group. Other studies have also reported this finding [22,29,32]. Apart from inflammatory markers, necrosis was determined in the epithelial cells of the bladder tissue of the cyc group. ın recent studies, there was no information that cyclophosphamide causes necrosis in bladder epithelial cells. However, in a previous study, it was stated that cystitis caused by cyclophosphamide is characterized by detachment of the urothelium, edema, microvascular damage, and focal muscle necrosis [33].
In our study, no histopathological damage was observed except mild and moderate dilatation, congestion, and PMNL infiltration in the connective tissue of the TCYC group. Histopathological findings were associated with the TCYC group's MDA, tGSH, TNF-α, IL-1β, and ıl-6 levels and its toS and taS values. ıt is known that the effect of taxifolin against oxidative stress is due to its inhibitory effect on ROS production [12]. ıt has also been proven that taxifolin has anti-inflammatory, antitumor, antimicrobial, and antioxidant properties [11]. No previous studies have investigated the antioxidant and anti-inflammatory effects of taxifolin on the bladder. It has been documented that taxifolin protects liver tissue from oxidative damage by inhibiting the increase in mda and toS and decreasing the tGSH levels and taS values due to pazopanib, an anticancer drug [13]. another study reported that taxifolin protected optic nerve tissue against oxidative and inflammatory damage of the platinum-derived anticancer drug [34]. It has been argued that the protective effect of taxifolin is due to the inhibition of MDA, TNF-α, and IL-1β overproduction [35]. It has also been shown experimentally that taxifolin does not have a significant effect on the normal oxidant-oxidant balance in healthy tissue [21].
Taxifolin has also been shown to have cytoprotective properties via activating nuclear factor erythroid-2 related factor 2 (Nrf2). ın response to oxidative stress, Nrf2 stimulates the transcription of cytoprotective genes, which is regulated by the adaptor protein kelch-like ecH-associating protein 1 (Keap-1). Physiologically, Nrf2 is present at a low level and is maintained through ubiquitination mediated by keap-1. Through the Nrf2dependent pathway, taxifolin increases the production of phase ıı antioxidant and detoxifying enzymes, which had a critical protective effect against DNA oxidative damage. ımportantly, taxifolin enhances Heme oxygenase-1 expression by increasing Nrf2 cytoplasmic expression and nuclear translocation. taxifolin may increase the expression of the Nrf2 gene and its nuclear translocation through epigenetic changes [36,37].
Quercetin is also a flavonoid that exhibited anti-inflammatory, anti-apoptotic, anti-thrombotic, and antitumor properties. the administration of quercetin effectively reduced cyclophosphamide-induced toxicity in multiple organs, including the heart, bladder, gonads, and lungs, in experimental mice. two prior investigations in the literature found quercetin to be protective against cyclophosphamide-induced hemorrhagic cystitis. these studies indicated that quercetin could help protect the bladder against cyclophosphamide urotoxicity by lowering MDA levels in the bladder and raising antioxidant decreased GSH levels in the bladder, indicating quercetin's antioxidant activity [38,39].
as a result, cyclophosphamide caused oxidative and inflammatory damage in the bladder tissue of rats. Taxifolin attenuated cyclophosphamide-related oxidative and inflammatory bladder injury by inhibiting the increase in the oxidants and proinflammatory cytokines and decreasing the antioxidants. The protective effect of taxifolin against cyclophosphamide-induced bladder damage has also been demonstrated histopathologically. Our experimental results indicate that taxifolin may be useful in the treatment of bladder damage due to the use of cyclophosphamide. However, histopathological studies at the molecular level are required to clarify the protective effect mechanism of taxifolin against cyclophosphamide-induced bladder toxicity. Furthermore, it is important to investigate the role of anti-inflammatory cytokines in the protective action mechanism of taxifolin.