Research ArticleExperimental Studies
Open Access

Oxysterol-binding Protein-like 3 Promotes Tumor Progression by Regulating Apoptosis and Angiogenesis in Colorectal Cancer

CHAN-MUK IM, HYUNG-HOON OH, SUN-YOUNG PARK, YOUNG-LAN PARK, JAE-SUNG KIM and YOUNG-EUN JOO
Cancer Genomics & Proteomics November 2025, 22 (6) 1025-1043; DOI: https://doi.org/10.21873/cgp.20553

Abstract

Background/Aim: Oxysterol-binding protein-like 3 (OSBPL3) is a member of the intracellular lipid receptor and transporter protein family involved in regulating lipid metabolism. Altered OSBPL3 expression has been observed in various cancers, where it has been associated with both oncogenic and tumor-suppressive roles. However, its precise functions and underlying mechanisms in colorectal cancer (CRC) remain unclear. This study aimed to investigate the role of OSBPL3 in CRC cells and evaluate its prognostic significance.

Materials and Methods: A small interfering RNA vector targeting OSBPL3 was employed to silence its expression in CRC cell lines. OSBPL3 levels in CRC tissues were assessed using reverse transcription-polymerase chain reaction and immunohistochemistry. Tumor cell apoptosis, proliferation, and angiogenesis were evaluated via a terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay, and immunohistochemical staining for Ki-67 and CD34.

Results: OSBPL3 silencing inhibited tumor cell migration and invasion in CRC. OSBPL3 knockdown reduced proliferation and induced apoptosis through caspase activation and cell cycle arrest mediated by the regulation of cyclins, cyclin dependent kinases (CDKs), and CDK inhibitors. OSBPL3 suppression impaired the invasion and tube formation of human umbilical vein endothelial cells by down-regulating angiogenic factors and up-regulating angiostatic factors. OSBPL3 expression correlated significantly with tumor size, tumor stage, invasion depth, lymph node involvement, distant metastasis, and reduced survival. The apoptotic index was lower and microvessel density was higher in OSBPL3-positive tumors than in OSBPL3-negative tumors.

Conclusion: OSBPL3 contributes to CRC progression by regulating tumor cell apoptosis and angiogenesis.

Keywords:

Introduction

Colorectal cancer (CRC) is among the most prevalent cancers and a leading cause of cancer-related mortality worldwide. Although evidence shows that the 5-year survival rate exceeds 90% when CRC is detected and treated early, most patients are still diagnosed at advanced stages, which are associated with poor prognosis (1-3). Therefore, since early detection and treatment can remarkably improve clinical outcomes, awareness of the signs, symptoms, and risk factors of CRC and the importance of routine screening, such as fecal occult blood tests and colonoscopy, should be raised (4-6).

CRC arises from the interplay of multiple factors, including genetic and epigenetic alterations, environmental carcinogen exposure, western diet, physical inactivity, alcohol use, smoking, and obesity (1-6). Obesity is a known risk factor for various cancers, including CRC, and it is closely associated with metabolic disturbances such as insulin resistance, mild hyperglycemia, dyslipidemia, and hypercholesterolemia, which may contribute to cancer initiation and progression (7, 8).

Oxysterols, which are oxygenated derivatives of cholesterol, are produced via the enzymatic activity of the cytochrome P450 family and non-enzymatic reactions involving reactive oxygen species. They have diverse biological effects depending on their concentration; they participate not only in cholesterol, steroid, and lipid metabolism and synthesis but also in the carcinogenesis and progression of several cancers (9-13).

Through a search for binding proteins possibly linking oxysterol levels to biological responses, a murine cytosolic protein that binds a range of oxysterols has been identified. This protein became the founding member of oxysterol-binding proteins (OSBPs) and their homologs, known as OSBP-like proteins (OSBPLs). OSBPs and OSBPLs form a large family of lipid-binding and lipid-transfer proteins. OSBPLs are implicated in cytoskeletal organization and cell adhesion, and they are also associated with cell proliferation, migration, and carcinogenesis (14-19). One of the members of this intracellular lipid receptor and transporter family is OSBPL3, which regulates lipid metabolism. Its expression is altered in various cancers and has been linked to oncogenic and tumor-suppressive roles (20-29). OSBPL3 is overexpressed in several cancers, including hepatocellular carcinoma, pancreatic cancer, gastric cancer, and CRC (20-27). Conversely, it is down-regulated in bladder cancer and B-cell lymphoma, where it likely functions as a tumor suppressor (28, 29). However, its precise role and underlying mechanisms in CRC development and progression remain unclear.

This study aimed to assess whether OSBPL3 influences oncogenic behaviors in CRC cell lines and tissues and to investigate its association with tumor cell survival, angiogenesis, and prognosis.

Materials and Methods

Cell culture and OSBPL3 siRNA transfection. Human CRC cell lines DLD1, HCT116, SW480, Caco-2, HT-29, and SW1116 (American Type Culture Collection, Manassas, VA, USA) were maintained in high-glucose Dulbecco’s modified Eagle medium (Hyclone, Logan, UT, USA) supplemented with 10% fetal bovine serum (Gibco, Thermo Fisher Scientific, Inc., Waltham, MA, USA) and 1% penicillin–streptomycin (Gibco, Thermo Fisher Scientific, Inc.). Cultures were incubated in a humidified chamber with 5% CO2 at 37°C. OSBPL3 siRNA (RNA-CGAAGCCAUUUAAUCCGGU; Bioneer, Daejeon, Republic of Korea) and scramble siRNA (Qiagen, Germantown, MD, USA) were transfected using Lipofectamine™ RNAiMAX (Invitrogen, Carlsbad, CA, USA) for 48 h in accordance with the manufacturer’s guidelines. For transient knockdown experiments, DLD1 and SW480 cells were seeded at a density of 3×106 cells per well in six-well plates. They were transfected with 100 pmol/well of siRNA and 5 μl of Lipofectamine RNAiMAX (Invitrogen). Afterward, they were incubated at 37°C in 5% CO2 until in vitro assays were initiated. Human umbilical vein endothelial cells (HUVECs) were acquired from Lonza (Walkersville, MD, USA) and cultured in an EBM™-2 basal medium + EGM™-2 SingleQuots™ supplements (Lonza, Basel, Switzerland).

Cell proliferation assessment. Cell proliferation was assessed using an EZ-CyTox WST-1 assay kit (Daeil Lab Inc., Seoul, Republic of Korea). The transfected cells were plated in 96-well plates and exposed to the WST-1 reagent at 37°C. Their proliferative activity was evaluated for 72 h, and absorbance was measured at 24, 48, and 72 h. Optical density was measured at 450 nm by using the SpectraMax® i3x Multi-Mode microplate reader (Molecular Devices, San Jose, CA, USA). Each experiment was performed in triplicate and independently repeated three times.

Protein extraction and western blotting. OSBPL3 siRNA-transfected cells were washed with phosphate-buffered saline (PBS) and lysed using Pierce™ RIPA buffer (Thermo Fisher Scientific, Inc.) supplemented with Halt™ phosphatase and protease inhibitor cocktails (Thermo Fisher Scientific, Inc.). The protein concentrations in the resulting lysates were quantified using a BCA protein assay kit (Thermo Fisher Scientific, Inc.). Equal amounts of protein were separated via SDS-PAGE on 8-12% polyacrylamide gels and electrotransferred onto Immobilon®-P PVDF membranes (Millipore, Billerica, MA, USA). The membranes were blocked with 5% BSA at room temperature for 1 h and incubated at 4°C overnight with primary antibodies diluted at 1:1,000. Immunoblotting was performed using antibodies against the following; OSBPL3 (Novus Biologicals, Centennial, CO, USA); β-tubulin and GAPDH (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA); and cleaved caspases 3 and 9, Poly(ADP-ribose) polymerase (PARP), Bcl-xL, cyclin B1, cyclin-dependent kinase (CDK) 4, p27, vimentin, E-cadherin, matrix metalloproteinase (MMP) 2, MMP9, snail, slug, hypoxia-inducible factor (HIF)-1α, vascular endothelial growth factor (VEGF)-A, -D, angiostatin, phospho-Akt, phospho-extracellular signal-regulated kinase (ERK)1/2, phospho-c-Jun N-terminal kinase (JNK), and phospho-p38 (Cell Signaling Technology, Inc., Danvers, MA, USA). After incubation with primary antibodies, the membranes were washed four times with Tris-buffered saline with 0.1% Tween-20 and incubated with horseradish peroxidase-conjugated secondary antibodies (Cell Signaling Technology) diluted at 1:2,000 for 1 h at room temperature. Protein bands were visualized using enhanced chemiluminescence (ECL, Millipore) and captured using the LAS-4000 imaging system (Fujifilm, Tokyo, Japan).

Flow cytometry analysis of apoptosis and cell cycle arrest. For flow cytometry analysis, siRNA-transfected cells were harvested through trypsinization and washed twice with PBS. Apoptotic cells were stained with APC-conjugated Annexin V and 7-AAD (BD Biosciences, San Diego, CA, USA). For cell cycle analysis, cell pellets were rinsed with cold PBS, fixed in 70% ice-cold ethanol, and washed again with PBS. The fixed cells were resuspended in staining buffer containing RNase A (10 μg/ml) and propidium iodide (50 μg/ml; Sigma-Aldrich, St. Louis, MO, USA). Samples were analyzed using a FACS-Calibur flow cytometer (Becton Dickinson, San Jose, CA, USA) and CellQuest software. Flow cytometry data were processed with WinMDI v2.9.

Wound healing assay. The migratory capacity of the transfected cells was evaluated using Ibidi culture inserts (Ibidi, Regensburg, Germany). A total of 5×104 transfected cells were seeded into each compartment of the insert and incubated at 37°C under humidified conditions for 24 h. After incubation, the inserts were carefully removed to create a defined, cell-free wound gap. Wound closure was monitored by imaging the gap at 32 and 54 h postinsert removal. Migratory efficiency was quantified by measuring the average reduction in the wound width across three randomly selected microscopic fields, and values were normalized to a 1 cm scale. The wound healing assay was performed in three independent experiments.

Cell invasion assay. Cell invasive potential was assessed using Transwell inserts with 8 μm pores (Corning, NY, USA). The upper surface of each insert was coated with 1% gelatin in a serum-free medium and incubated at 37°C overnight. The transfected cells were suspended in 100 μl of serum-free medium containing 0.2% (w/v) bovine serum albumin (BSA; Sigma-Aldrich) and added to the upper chamber. The lower chamber was filled with 400 μl of 0.2% BSA supplemented with 20 μg/ml human plasma fibronectin (Merck KGaA, Darmstadt, Germany). The assembled chambers were incubated in a humidified environment containing 5% CO2 for 24 h. The cells that invaded through the membrane were fixed with 70% ethanol and stained with Hemacolor® Rapid staining solution (Merck) at room temperature. The number of invading cells on the lower membrane surface was quantified by counting the stained cells in six randomly selected microscopic fields. The invasion assay was carried out in three independent experiments.

Matrigel tube formation assay. In this procedure, 96-well tissue culture plates were pre-coated with Matrigel (10 mg/ml; BD Biosciences) to evaluate the angiogenic potential. HUVECs were resuspended in a conditioned medium, defined as the culture supernatant from OSBPL3 knockdown cells incubated in a serum-free medium, and seeded onto a polymerized Matrigel surface. After they were incubated overnight, the formation of capillary-like tubular networks was observed and imaged using an inverted phase-contrast microscope. The total tube length was quantitatively analyzed using the WIMtube image analysis software (WIMASIS GmbH, Munich, Germany). The tube formation assay was repeated in three independent experiments.

Matrigel invasion assay. For endothelial cell invasion assays, Transwell chambers with 8-μm pore filters were coated with Matrigel (1 mg/ml; BD Biosciences) and air-dried at room temperature. HUVECs were seeded in duplicate into the upper chambers at a density of 3×104 cells per well in serum-free EGM-2 medium. The lower chambers were filled with the conditioned medium (CM) collected from OSBPL3 knockdown cells cultured in serum-free conditions. After 3 h of incubation at 37°C, the cells that migrated through the Matrigel and adhered to the underside of the membrane were fixed and stained using a Diff-Quik solution (Sysmex, Kobe, Japan). The Matrigel invasion assay was independently repeated three times.

Patient population and tissue preparation. Fresh tumors and matched adjacent normal colorectal tissues were obtained from 20 patients undergoing endoscopic biopsy at Chonnam National University Hwasun Hospital (Jeonnam, Republic of Korea). Paraffin-embedded tissues and clinical data were collected from 261 consecutive patients who had CRC and underwent surgery at the same hospital between January and December 2016 to investigate the prognostic relevance of OSBPL3 expression. None of the patients received preoperative chemotherapy or radiotherapy. Tumor staging was determined according to the American Joint Committee on Cancer (AJCC) TNM classification (30). Patient survival was monitored from the date of surgery until December 31, 2023. The study protocol was approved by the Institutional Review Board of Chonnam National University Hwasun Hospital (IRB No. CNUHH-2025-125) and written informed consent including permission to store and utilize clinical data for research purposes, was obtained from all participants.

Reverse transcription-polymerase chain reaction. Total RNA was extracted from fresh tissue specimens by using TRIzol reagent (Invitrogen). Complementary DNA (cDNA) was synthesized using MMLV reverse transcriptase and then amplified via PCR with gene-specific primers and GoTaq® DNA polymerase (Promega, Madison, WI, USA). The following primer sequences were used: OSBPL3 (forward: 5′-CAGTCTTCAGAGGACATGGA-3′; reverse: 5′-TAGACTC TCCATCACTGACT-3′) and GAPDH (forward: 5′-CACAG TCCATGCCATCAC-3′; reverse: 5′-CACCACCCTGTTGCTGTA-3′). PCR products were resolved via agarose gel electrophoresis and visualized using a HiQ BlueMango staining solution (BioD Co., Ltd, Gyeonggi-do, Republic of Korea).

Immunohistochemical staining. The paraffin-embedded tissue sections were deparaffinized, rehydrated, and subjected to heat-induced antigen retrieval. Endogenous peroxidase activity was quenched, and the sections were incubated with primary antibodies against OSBPL3 (Novus Biologicals), Ki-67 (Abcam, Cambridge, UK), and CD34 (Abcam) at 4°C overnight. Detection was conducted using the Dako Real™ Envision HRP/DAB system, followed by hematoxylin counterstaining. The stained sections were examined and imaged using a standard light microscope.

Evaluation of OSBPL3 expression. Immunohistochemical staining was independently evaluated by two blinded observers. The staining intensity and the percentage of positive tumor cells were scored on a four-point scale. The final immunoreactivity score was calculated as the product of the two scores. A mean score of ≥6.0 was used to define a high OSBPL3 expression, while scores below this threshold indicated a low expression.

Assessment of tumor cell proliferation and microvessel density (MVD). Tumor cell proliferation was assessed by Ki-67 immunostaining; the Ki-67 labeling index (KI) was defined as the number of Ki-67-positive nuclei per 1,000 tumor cells. MVD was determined via CD34 immunostaining. Any discrete CD34-positive endothelial cell or cluster clearly demarcated from the surrounding structures was considered a microvessel. Vessels were counted in five high-density vascular regions at 200× magnification, and the mean value was recorded as the MVD.

Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay and determination of the apoptotic index (AI). Apoptotic cells were detected using the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay system (Promega). Formalin-fixed and paraffin-embedded sections were deparaffinized in xylene and rehydrated through a graded ethanol series. They were permeabilized by treating them with proteinase K buffer to enhance DNA accessibility. The TdT enzyme reaction mixture was applied to enzymatically label DNA strand breaks with modified nucleotides. After the cells were washed, chromogenic detection was performed using 3,3′-diaminobenzidine (DAB) to visualize TUNEL-positive nuclei. The AI was calculated as the number of TUNEL-positive nuclei per 1,000 tumor cells.

Statistical analysis. Groups were compared using Student’s t-test, and results were presented as mean±standard deviation (SD). The associations between the OSBPL3 expression and clinicopathological parameters were evaluated using the χ2 test or Fisher’s exact test as appropriate. Survival curves were constructed using the Kaplan-Meier method and compared via the log-rank test. All statistical analyses were conducted using IBM SPSS version 22.0 (Armonk, NY, USA), and data with p-values less than 0.05 were considered statistically significant.

Results

Expression of OSBPL3 in CRC cells. Western blotting was performed to detect the OSBPL3 protein levels in several CRC cell lines, including DLD1, HCT116, SW480, Caco-2, HT-29, and SW1116, and to assess the OSBPL3 expression in CRC cells. The results showed that the OSBPL3 expression was notably elevated in DLD1 and SW480 cells (Figure 1A). Accordingly, OSBPL3 siRNA was utilized to knock down the endogenous OSBPL3 expression in DLD1 and SW480 cells and explore the role of OSBPL3 in the oncogenic behavior of CRC cells. The OSBPL3 protein levels were markedly reduced following siRNA transfection (Figure 1B).

Figure 1.
Figure 1.

Expression of OSBPL3 in CRC cells. (A) Expression of OSBPL3 protein in human CRC cell lines, including DLD1, HCT116, SW480, Caco-2, HT-29, and SW1116. (B) Expression of OSBPL3 protein following transfection with scramble siRNA or OSBPL3 siRNA in DLD1 and SW480 cells. Western blot analysis confirmed the effective knockdown of OSBPL3 protein by using Si-O3. OSBPL3, Oxysterol-binding protein-like 3; CRC, colorectal cancer; SS, scramble siRNA; Si-O3, OSBPL3 siRNA.

Effect of OSBPL3 knockdown on proliferation, apoptosis, and cell cycle of CRC cells. To assess the effect of OSBPL3 knockdown on cell proliferation, a proliferation assay was conducted at 1, 2, and 3 days following OSBPL3 siRNA transfection. Proliferation, measured by absorbance, was significantly reduced in OSBPL3 siRNA-transfected DLD1 and SW480 cells compared to scramble siRNA-transfected controls at day 3 (p<0.001 for both; Figure 2A). To evaluate whether OSBPL3 knockdown-induced apoptosis, flow cytometry analyses were performed. Apoptotic rates were notably higher in OSBPL3 siRNA transfected DLD1 and SW480 cells than in the scramble controls (10.3% vs. 14.8% and 9.8% vs. 15.3%, respectively; Figure 2B). The caspase-specific activity was examined to assess caspase activation during OSBPL3 knockdown-induced apoptosis. The expression levels of cleaved caspase-3, -9, and PARP were up-regulated in DLD1 and SW480 cells after OSBPL3 knockdown (Figure 2C). The Bcl-xL protein expression was down-regulated by OSBPL3 knockdown in both cell lines (Figure 2C). Flow cytometry analyses were also conducted to determine whether OSBPL3 influences cell cycle distribution. OSBPL3 knockdown induced cell cycle arrest at the subG1 phase in DLD1 and SW480 cells (Figure 2D). The effect of OSBPL3 on CDK inhibitors (CDKIs) involved in cell cycle regulation was further investigated. The p27 protein level was markedly increased following OSBPL3 knockdown (Figure 2E). CDKIs negatively regulate cyclins and CDKs; therefore, the expression levels of cyclin B1 and CDK4 were examined. The results showed that OSBPL3 knockdown significantly reduced the cyclin B1 and CDK4 protein levels in DLD1 and SW480 cells (Figure 2E).

Figure 2.
Figure 2.
Figure 2.

Effect of OSBPL3 knockdown on the proliferation, apoptosis, and cell cycle of CRC cells. (A) Effect of OSBPL3 knockdown on the proliferation of CRC cells. Cell viability, as indicated by absorbance, significantly decreased in Si-O3-transfected DLD1 and SW480 cells on day 3 (mean±SD, n=3; *p<0.05). (B) Effect of OSBPL3 knockdown on the apoptosis of CRC cells. The proportion of apoptotic cells was increased following Si-O3 transfection compared with SS (10.3% vs. 14.8% and 9.8% vs. 15.3% in DLD1 and SW480 cells, respectively). (C) Expression levels of caspases and apoptosis-related proteins. The levels of cleaved caspase-3, -9, and PARP increased after OSBPL3 knockdown in DLD1 and SW480 cells. The expression of the anti-apoptotic protein Bcl-xL decreased. (D) Effect of OSBPL3 knockdown on cell cycle distribution. Knockdown caused the accumulation of DLD1 and SW480 cells in the subG1 phase. Data represent one of three independent experiments. (E) Expression levels of cyclins, CDKs, and CDK inhibitors. OSBPL3 knockdown significantly reduced the cyclin B1 and CDK4 levels and increased the p27 expression in DLD1 and SW480 cells. OSBPL3, Oxysterol-binding protein-like 3; CRC, colorectal cancer; SS, scramble siRNA; Si-O3, OSBPL3 siRNA; SD, standard deviation; PARP, poly (ADP-ribose) polymerase; CC, cleaved caspase; CDK, cyclin-dependent kinase.

Effect of OSBPL3 knockdown on the migration and invasion of CRC cells. The artificial wound gap in scramble siRNA-transfected cells became significantly narrower than that in OSBPL3 siRNA-transfected cells at 54 h in DLD1 and SW480 cells (p<0.001 and p=0.028, respectively; Figure 3A). A total of 52.0±145.6 and 8±3.7 invading DLD1 and SW480 cells transfected with OSBPL3 siRNA were respectively detected; by comparison, 893.3±167.4 and 54.0±11.2 invading DLD1 and SW480 cells transfected with scramble siRNA were found. These values were determined by averaging the counts from six randomly selected squares (0.5×0.5 mm2 microscope fields) with 10 μg/ml fibronectin as a chemoattractant. They were significantly different in DLD1 and SW480 cells (p=0.022 and p<0.001, respectively; Figure 3B). OSBPL3 knockdown reduced the expression levels of vimentin, MMP-2, MMP-9, snail, and slug and increased the expression level of E-cadherin in DLD1 and SW480 cells (Figure 3C).

Figure 3.
Figure 3.
Figure 3.

Effect of OSBPL3 on the migration and invasion of CRC cells. (A) Representative images and quantitative graphs illustrating the effect of OSBPL3 knockdown on CRC cell migration. A wound healing assay was performed using siRNA-transfected cells, and relative healing distances were presented as mean±SD (n=3; *p<0.05). The artificial wound gap in SS-transfected cells became significantly narrower than that in Si-O3-transfected cells at 54 h in DLD1 and SW480 cells. (B) Representative images with corresponding quantitative graphs demonstrating the effect of OSBPL3 knockdown on CRC cell invasion. An invasion assay was conducted using siRNA-transfected cells. Invading cells were stained, counted, and graphed for each group. The number of invading Si-O3-transfected cells was significantly lower than that of SS-transfected DLD1 and SW480 cells (mean±SD, n=3; *p<0.05). (C) Representative images and quantitative data showing the effect of OSBPL3 knockdown on extracellular matrix-related proteins. OSBPL3 knockdown reduced the expression levels of vimentin, MMP-2, MMP-9, snail, and slug and increased the expression of E-cadherin in DLD1 and SW480 cells. OSBPL3, Oxysterol-binding protein-like 3; CRC, colorectal cancer; SS, scramble siRNA; Si-O3, OSBPL3 siRNA; SD, standard deviation; MMP, matrix metalloproteinase.

Effect of OSBPL3 knockdown on the angiogenesis of CRC cells. Matrigel invasion and tube formation assays were performed using the conditioned media (CM) from OSBPL3 or scramble siRNA-transfected DLD1 and SW480 cells to assess the effect of OSBPL3 on HUVEC angiogenesis. HUVEC invasion was significantly decreased when the CRC cells were cultured in the CM from OSBPL3 siRNA-transfected cells compared with those in the CM from scramble controls (p=0.019 and p=0.031, respectively; Figure 4A). Moreover, the CM from OSBPL3 siRNA-transfected DLD1 and SW480 cells markedly inhibited endothelial tube formation compared with that in the CM from scramble siRNA-transfected cells (p=0.043 and p=0.013, respectively; Figure 4B). OSBPL3 knockdown also suppressed the expression of the angiogenic inducers HIF-1α, VEGF-A, and VEGF-D and increased the expression of the angiogenesis inhibitor angiostatin in DLD1 and SW480 cells (Figure 4C).

Figure 4.
Figure 4.

Effect of OSBPL3 knockdown on the angiogenesis of CRC cells. (A) Representative images and statistical analyses depicting the effect of OSBPL3 knockdown on HUVECs invasion. The invasion of HUVECs cultured in CM from Si-O3-transfected DLD1 and SW480 cells significantly decreased compared to that of HUVECs cultured in CM from SS-transfected cells (p=0.019 and p=0.031, respectively). (B) Representative images alongside quantitative graphs presenting the effect of OSBPL3 knockdown on endothelial tube formation. CM from Si-O3-transfected DLD1 and SW480 cells significantly inhibited endothelial tube formation compared with CM from SS-transfected cells (p=0.043 and p=0.013, respectively). (C) Representative images and quantitative graphs demonstrating the effect of OSBPL3 knockdown on the expression of angiogenesis-related proteins. OSBPL3 knockdown decreased the expression levels of the pro-angiogenic factors HIF-1α, VEGF-A, and VEGF-D and increased the expression of the anti-angiogenic factor angiostatin in DLD1 and SW480 cells. OSBPL3, Oxysterol-binding protein-like 3; CRC, colorectal cancer; SS, scramble siRNA; Si-O3, OSBPL3 siRNA; CM, conditioned media; HUVECs, human umbilical vein endothelial cells; HIF-1α, Hypoxia-inducible factor-1α; VEGF, vascular endothelial growth factor. Each experiment was repeated three times. *p<0.05 vs. control.

Effect of OSBPL3 knockdown on oncogenic signaling pathways involved in the regulation of OSBPL3 expression in CRC cells. To investigate whether OSBPL3 activates intracellular signaling pathways in CRC cells, we assessed the phosphorylation levels of Akt and MAPKs signaling proteins via western blotting. OSBPL3 knockdown led to reduce the phosphorylation of Akt and JNK, while the phosphorylation levels of ERK1/2 and p38 remained unchanged (Figure 5).

Figure 5.
Figure 5.

Effect of OSBPL3 knockdown on oncogenic signaling pathways involved in the regulation of the OSBPL3 expression in CRC cells. The phosphorylation levels of Akt and JNK were decreased following OSBPL3 knockdown, whereas the phosphorylation levels of ERK1/2 and p38 remained unchanged. OSBPL3, Oxysterol-binding protein-like 3; CRC, colorectal cancer; SS, scramble siRNA; Si-O3, OSBPL3 siRNA; ERK1/2, extracellular signal-regulated kinase1/2; JNK, c-Jun N-terminal kinase.

Expression of OSBPL3 in CRC and metastatic lymph node tissues. To validate the findings from CRC cell lines, we examined the OSBPL3 expression at RNA and protein levels via RT-PCR, western blotting, and immunohistochemistry in CRC tissues, paired normal colorectal mucosa, and metastatic or non-metastatic lymph node tissues obtained via colonoscopic biopsy and surgical specimens from the same patients. In colonoscopic biopsy samples, the OSBPL3 expression was significantly up-regulated at the RNA level in cancer tissues compared with that in the paired normal mucosa (p<0.001; Figure 6A). In paraffin-embedded tissue sections, OSBPL3 protein showed weak or no staining in the normal colorectal mucosa (Figure 6B). Conversely, OSBPL3 immunostaining was predominantly observed in the cytoplasm of cancer cells, but a signal was not detectable in the tumor stroma (Figure 6B). Furthermore, the OSBPL3 expression in metastatic lymph node tissues was significantly stronger than that in non-metastatic nodes (Figure 6C). The overall immunostaining score of OSBPL3 was markedly higher in metastatic lymph nodes than in non-metastatic counterparts (p<0.001; Figure 6D).

Figure 6.
Figure 6.
Figure 6.

Expression of OSBPL3 in CRC and metastatic lymph node tissues. (A) Expression images and band quantification graphs of OSBPL3 mRNA levels in colorectal cancer tissue and paired normal mucosa by reverse-transcription PCR. The mRNA expression of OSBPL3 was up-regulated in cancer tissues compared with that in the paired normal mucosa in fresh colonoscopic biopsy specimens. Each bar represents the mean±SD of 20 cases. *p<0.05 vs. normal colorectal mucosa. (B) Immunohistochemical staining of OSBPL3 in normal colorectal mucosa and colorectal cancer tissue. The immunohistochemical staining of OSBPL3 protein showed weak or no staining in normal colorectal mucosa. Conversely, cancer cells exhibited strong cytoplasmic staining, but the tumor stroma had no detectable staining (original magnification, ×100 and ×400). (C) Immunohistochemical staining of OSBPL3 in non-metastatic lymph node and metastatic lymph node (original magnification: ×400). OSBPL3 immunostaining in metastatic lymph nodes was significantly stronger than that in non-metastatic lymph nodes. (D) The overall immunostaining score was significantly higher in metastatic lymph node tissues than in non-metastatic lymph node tissues (*p<0.05; original magnification: ×400). OSBPL3, Oxysterol-binding protein-like 3; CRC, colorectal cancer; T; colorectal cancer tissue, N; paired normal colorectal mucosa, NL; non-metastatic lymph node, ML; metastatic lymph node.

Correlation between the OSBPL3 expression and clinicopathological features in CRCs. To examine the prognostic significance of OSBPL3 in CRC progression, we immunohistochemically assessed the OSBPL3 protein expression in formalin-fixed and paraffin-embedded tissue blocks from 261 CRC patients with available clinicopathological data, including survival. The correlation between OSBPL3 immunostaining and clinicopathological features was also analyzed. OSBPL3 immunostaining was significantly associated with tumor size, tumor stage, invasion depth, lymph node involvement, and distant metastasis (p=0.002, p<0.001, p=0.022, p<0.001, and p<0.001, respectively; Table I). Moreover, the overall survival was significantly lower in patients with positive OSBPL3 immunostaining than in patients without positive OSBPL3 immunostaining (p=0.004; Figure 7).

View this table:
Table I.

Correlation of the OSBPL3 expression with the clinicopathological parameters of patients with colorectal cancer.

Figure 7.
Figure 7.

Kaplan-Meier survival curve correlating overall survival with the negative (solid line) and positive expression (dotted line) of OSBPL3. OSBPL3, Oxysterol-binding protein-like 3.

Correlation of OSBPL3 expression with tumor cell apoptosis, proliferation, or angiogenesis in CRCs. All tumor samples were subjected to TUNEL assay and immunostaining for Ki-67 and CD34 to evaluate tumor cell apoptosis, proliferation, and angiogenesis (Figure 8). The KI in the 261 tumors ranged from 32.6 to 89.3 (mean of 61.2±16.4). The OSBPL3 expression was not significantly associated with KI (p=0.592). The AI ranged from 4.7 to 17.2 (mean of 8.6±6.2). The mean AI of OSBPL3-positive tumors was 7.1±4.1, which was significantly lower than that of OSBPL3-negative tumors (p=0.031). The MVD ranged from 3.5 to 429.0 (mean of 108.4±96.5). The mean MVD in OSBPL3-positive tumors was 122.3±81.4, which was significantly higher than that in OSBPL3-negative tumors (p=0.025; Table II).

Figure 8.
Figure 8.

Assessment of tumor cell apoptosis, proliferation, and angiogenesis in CRCs. (A) Detection of apoptotic cells and bodies by TUNEL staining. (B) Square box in A. Apoptotic bodies were identified by the presence of pyknotic nuclei surrounded by a condensed cytoplasm and separated from neighboring cells by a clear halo. (C) Immunohistochemical staining of Ki-67 in CRC tissue. (D) Square box in C. Ki-67 immunoreactivity was observed in the nuclei of proliferating cancer cells. (E) Immunohistochemical staining of CD34 in CRC tissue. (F) Square box in D. Antibody-specific vessels were defined as single brown-stained cells or clusters of endothelial cells clearly distinguishable from adjacent microvessels and tumor cells (scale bar corresponds to 10 μm). OSBPL3, Oxysterol-binding protein-like 3; CRC, colorectal cancer; TUNEL, terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick end labeling.

View this table:
Table II.

Correlation of the OSBPL3 expression with tumor cell apoptosis, proliferation and angiogenesis in colorectal cancer tissues.

Discussion

OSBPL3 is an intracellular lipid receptor and transporter belonging to the OSBP superfamily, which regulates cell adhesion, lipid metabolism, vesicular transport, and cytoskeletal organization under physiological conditions. Its expression is altered in various cancers, in which it may function as either an oncogene or a tumor suppressor in a cancer type-specific manner (20-29). However, its specific role and underlying mechanisms in CRC development and progression remain unclear.

The colorectal mucosa undergoes a dynamic cycle of continuous proliferation, differentiation, apoptosis, and cell cycle regulation. These processes should be properly controlled to maintain homeostasis and tissue architecture in the colorectal epithelium (31, 32). However, dysregulation provides survival advantages to CRC cells and facilitates tumor invasion and metastasis (33-36). This process involves the following key events: the proteolysis of the extracellular matrix (ECM) surrounding the tumor (33) and the epithelial-mesenchymal transition (EMT) through which cells lose their epithelial characteristics and acquire mesenchymal traits (34). We initially investigated whether OSBPL3 knockdown influences tumor cell migration, invasion, and survival in CRC cells. Our results demonstrated that OSBPL3 knockdown inhibited cell migration and invasion by down-regulating ECM-degrading proteolytic enzymes (MMPs) and mesenchymal markers (vimentin, snail, and slug) and up-regulating the epithelial marker E-cadherin. Additionally, OSBPL3 knockdown suppressed proliferation and induced apoptosis by activating caspase-specific pathways and causing cell cycle arrest through the down-regulation of cyclins and CDKs and the up-regulation of CDK inhibitors. Previously, OSBPL3 overexpression was shown to promote the proliferation, invasion, and metastasis of CRC cells in vitro and in vivo using a subcutaneous xenograft model (26). Therefore, OSBPL3 potentially regulates CRC cell migration, invasion, and survival.

Tumor angiogenesis refers to the formation of new blood vessels to supply nutrients and oxygen to growing tumors. It is essential for tumor growth and metastasis because it facilitates the entry of tumor cells into the bloodstream; thus, they can spread to distant sites (37-39). A growing body of evidence from in vitro and in vivo studies has demonstrated that CRC progression is heavily dependent on tumor angiogenesis (40, 41). Previous studies revealed that certain OSBP family members, such as OSBPL2 and OSBPL7, participate in lipid homeostasis, metabolism, and angiogenic signaling in endothelial cells (42-44). On this basis, we examined the effect of OSBPL3 expression on angiogenic phenotypes via in vitro assays in CRC cells. We found that OSBPL3 knockdown significantly reduced the invasion and tube formation of HUVECs. Furthermore, OSBPL3 knockdown down-regulated angiogenic factors, HIF-1α, VEGF-A and VEGF-D and up-regulated the angiogenesis inhibitor, angiostatin in CRC cells. These findings suggested that OSBPL3 might contribute to tumor progression by promoting angiogenesis through its interaction with pro- and anti-angiogenic factors in CRC. Therefore, our CRC cell line studies indicated that OSBPL3 contributed to the acquisition of invasive phenotypes and functioned as an oncogenic factor in CRC.

To investigate the potential mechanisms underlying the effects of OSBPL3, we examined its influence on multiple intracellular signaling pathways involved in tumor cell migration, invasion, survival, and angiogenesis. Akt and mitogen-activated protein kinase (MAPK) signaling pathways regulate motility, adhesion, proliferation, survival, EMT, and angiogenesis in various cancer types (45-48). In our study, OSBPL3 knockdown led to decreased phosphorylation levels of Akt and JNK, whereas the phosphorylation levels of ERK1/2 and p38 remained unchanged. In a previous study, OSBPL3 promotes tumor growth by activating the R-Ras/Akt signaling pathway in gastric cancer cells (23).

We further examined the OSBPL3 expression in CRC tissues and paired normal colorectal mucosa from the same patients by using colonoscopic biopsy samples and in metastatic versus non-metastatic lymph node tissues collected during surgery. The OSBPL3 expression was significantly increased in CRC and metastatic lymph nodes compared with that in the normal mucosa and non-metastatic nodes. Additionally, we evaluated the OSBPL3 expression in CRC patients with long-term and complete follow-up and focused on its prognostic relevance. The positive OSBPL3 expression was significantly associated with tumor size, tumor stage, invasion depth, lymph node involvement, distant metastasis, and reduced overall survival. Previous studies similarly reported a high OSBPL3 expression in CRC tissues and its associations with KRAS mutations, poor differentiation, and unfavorable prognosis at mRNA and protein levels (25, 26). Conversely, another previous study found that lower OSBPL3 mRNA levels are correlated with a worse survival of CRC patients with advanced nodal metastasis and female patients with grade 3 tumors (27). Therefore, OSBPL3 might serve as a potential molecular target with a therapeutic value and act as a prognostic biomarker in CRC.

We evaluated the correlation of OSBPL3 expression with tumor cell apoptosis, proliferation, and angiogenesis in CRC tissues. We found that the mean AI was significantly lower and the MVD was significantly higher in OSBPL3-positive tumors compared to OSBPL3-negative tumors. Although a previous study reported that a high OSBPL3 expression is strongly related to increased Ki-67 levels (25), we did not observe a significant correlation between OSBPL3 and Ki-67 expression levels in our analysis. These findings suggest that the effects of OSBPL3 on tumor cell survival and angiogenesis observed in CRC cell lines are also reflected in human CRC tissues.

Conclusion

OSBPL3 potentially contributes to tumor progression by modulating apoptosis and angiogenesis in CRC. Therefore, it may serve as a promising biomarker and therapeutic target in CRC.

Acknowledgements

This study was supported by a grant (HCRI-25023) from the Chonnam National University Hwasun Hospital Institute for Biomedical Science, Republic of Korea, and partly by the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Education (RS-2023-00247166).

Footnotes

  • Conflicts of Interest

    The Authors declare that they have no conflicts of interest in regard to this study.

  • Authors’ Contributions

    CMI, HHO, YLP, SYP, JSK and YEJ conceived and designed the present study. CMI, HHO, YLP, SYP, JSK and YEJ performed the experiments. CMI, HHO and YEJ collected and analyzed the data. CMI, HHO, YLP, SYP, JSK and YEJ wrote, reviewed and/or revised the manuscript. All Authors read and approved the final manuscript.

  • Artificial Intelligence (AI) Disclosure

    No artificial intelligence (AI) tools, including large language models or machine learning software, were used in the preparation, analysis, or presentation of this manuscript.

  • Received August 9, 2025.
  • Revision received September 4, 2025.
  • Accepted September 20, 2025.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).

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Oxysterol-binding Protein-like 3 Promotes Tumor Progression by Regulating Apoptosis and Angiogenesis in Colorectal Cancer
CHAN-MUK IM, HYUNG-HOON OH, SUN-YOUNG PARK, YOUNG-LAN PARK, JAE-SUNG KIM, YOUNG-EUN JOO
Cancer Genomics & Proteomics Nov 2025, 22 (6) 1025-1043; DOI: 10.21873/cgp.20553
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